Novel polypeptide

ABSTRACT

Polynucleotide and polypeptide sequences are described. The polypeptide sequences comprise one or more of: (a) a polypeptide having the deduced amino acid sequence translated from the polynucleotide sequence in SEQ ID NO: 1 and variants, fragments, homologues, analogues and derivatives thereof; (b) a polypeptide of SEQ ID NO: 2 and variants, fragments, homologues, analogues and derivatives thereof; or (c) a polypeptide encoded by the cDNA of NCIMB 41066 and variants, fragments, homologues, analogues and derivatives thereof.

TECHNICAL FIELD

The present invention relates to a novel polynucleotide sequence whichencodes a novel polypeptide belonging to the class of proteins known asG-protein coupled receptors (GPCRs). The present invention also relates,inter alia, to processes for producing the polypeptide and its uses.

BACKGROUND OF THE INVENTION

Cells and tissues respond to a wide variety of extracellular signallingmolecules through the interaction of these molecules with specificcell-surface receptors. One such class of receptors are known asG-protein coupled receptors (GPCRs) and these are characterised bycontaining a series of 7 hydrophobic transmembrane segments. Uponbinding an extracellular ligand to its receptor, intracellular signalsare initiated via interactions with heterotrimeric G proteins which, inturn, can lead to a number of different intracellular events dependingupon which receptor has been activated. For example some GPCRs influenceadenyl cyclase activity whereas others act via phospholipase C.

Members of the GPCR superfamily respond to a wide variety of ligandsincluding small molecule amines (such as serotonin, dopamine,acetylcholine), lipid-derived mediators (such as LpA), amino acidderivatives (such as glutamate) and neurotransmitter peptides andhormones (such as neurokinin, galanin, glucagon, gastrin). AlthoughGPCRs are activated by a broad range of ligands, it should be noted thatindividual GPCRs have a small and very specific repertoire of ligands.Based upon an analysis of the primary structure of a novel GPCR, it isnow possible to classify them into specific sub-families, therebynarrowing the range of potential ligands.

In many cases, the endogenous ligands of GPCRs are relatively small,enabling them to be mimicked or blocked by synthetic analogues. Forexample drugs such as prazosin, doxazosin, cimetidine, ranitidine areall effective antagonists of their respective target GPCRs.

Thus, as the modulation of GPCRs can have therapeutic consequences,there is a continued need to provide new GPCRs and their associatedagonists and antagonists.

SUMMARY OF THE INVENTION

In a broad aspect, the present invention relates to novel amino acidsequences. In this regard, a specific novel amino acid sequence has beenisolated and it is to be understood that the invention covers thatsequence as well as novel variants, fragments, derivatives andhomologues thereof.

In another broad aspect, the present invention relates to novel nucleicacid sequences. In this regard, a specific novel nucleic acid sequencehas been isolated and it is to be understood that the invention coversthat sequence as well as novel variants, fragments, derivatives andhomologues thereof.

Thus, in brief, some aspects of the present invention relate to:

-   -   1. Novel amino acids.    -   2. Novel nucleotide sequences.    -   3. Assays using said novel sequences.    -   4. Compounds/compositions identified by use of said assays.    -   5. Expression systems comprising or expressing said novel        sequences.    -   6. Methods of treatment based on said novel sequences.    -   7. Pharmaceutical compositions based on said novel sequences.

Other aspects concerning the amino acid sequence of the presentinvention and/or the nucleotide sequence of the present inventioninclude: a construct comprising or capable of expressing the sequencesof the present invention; a vector comprising or capable of expressingthe sequences of the present invention; a plasmid comprising or capableof expressing the sequences of the present invention; a cell transfectedor virally-transduced with a construct/vector/plasmid comprising orcapable of expressing the sequences of the present invention; a tissuecomprising or capable of expressing the sequences of the presentinvention; an organ comprising or capable of expressing the sequences ofthe present invention; a transformed host comprising or capable ofexpressing the sequences of the present invention; and a transformedorganism comprising or capable of expressing the sequences of thepresent invention. The present invention also encompasses methods ofexpressing the same, such as expression in a micro-organism; includingmethods for transferring the same.

For ease of reference, aspects of the present invention are nowdiscussed under appropriate section headings. However, the teachingsunder each section are not necessarily limited to each particularsection.

In the following commentary references to “nucleotide sequence of thepresent invention” and “amino acid sequence of the present invention”refer respectively to any one or more of the nucleotide sequencespresented or discussed herein and to any one or more of the amino acidsequences presented or discussed herein. Also, and as used herein,“amino acid sequence” refers to peptide or protein sequences and mayrefer to portions thereof. In addition, the term “amino acid sequence ofthe present invention” is synonymous with the phrase “polypeptidesequence of the present invention”. Also, the term “nucleotide sequenceof the present invention” is synonymous with the phrase “polynucleotidesequence of the present invention”.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims. While the inventionis described in connection with specific embodiments, it will beunderstood that it is capable of further modifications. Therefore, thisapplication is intended to cover any variations, uses, or adaptations ofthe invention that follow, in general, the principles of the invention,including departures from the present disclosure that come within knownor customary practice within the art. All publications mentioned hereinare incorporated by reference in their entireties.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schema for the bioinformatic analysis of PFI-002(db=database).

FIG. 2 shows the specific functional response of PFI-002 receptor toneuromedin U. The grid corresponds to a 96 well plate and each square ofthe grid corresponds to one screened peptide (see list of screenedpeptides under FUNCTIONAL STUDIES section below—e.g. square A1 relatesto the result for peptide Arg-Gly-Asp-Ser (RGDS); square B1 relates tothe result for peptide [Arg8]Vasopressin, etc.).

FIG. 3 shows a ClustalW alignment of PFI-002 with SW|P20789|NTR1_RATNEUROTENSIN RECEPTOR TYPE 1 (NT-R-1).

FIG. 4 shows a ClustalW alignment of PFI-002 withGB|AF044601|NMUR1_Human NEUROMEDIN RECEPTOR TYPE 1 (NMU-R-1).

FIG. 5A shows SEQ ID NO: 1 which is the nucleotide sequence coding forPFI-002. The ATG translation initiation codon is indicated by the firstthree letters. The stop codon is indicated by the last three letters.

FIG. 5B shows SEQ ID NO: 2 shows the corresponding amino acid sequencecoding for PFI-002.

FIG. 6 shows SEQ ID NOS: 3-6 which are the various primers usedthroughout the Examples.

DETAILED DESCRIPTION

According to one aspect of the present invention, there is provided anisolated and/or purified polynucleotide comprising one or more of:

-   -   (a) a polynucleotide encoding the polypeptide as set forth in        SEQ ID NO: 2;    -   (b) a polynucleotide comprising a nucleotide sequence of SEQ ID        NO: 1;    -   (c) a polynucleotide encoding the polypeptide expressed by the        DNA contained in NCIMB 41066;    -   (d) a polynucleotide comprising a nucleotide sequence that has        at least 70% identity to the polynucleotide of any one of (a) to        (c);    -   (e) a polynucleotide comprising a nucleotide sequence which is        capable of hybridising to the polynucleotide of any one of (a)        to (d);    -   (f) a complement to the polynucleotide of any one of (a) to (e);        or    -   (g) a polynucleotide fragment of the polynucleotide of any one        of (a) to (f).

Preferably, the polynucleotide comprises a nucleotide sequence that hasat least 75% identity to the polynucleotide of any one of (a) to (c).More preferably, the polynucleotide comprises a nucleotide sequence thathas at least 80% identity to the polynucleotide of any one of (a) to(c). Even more preferably, the polynucleotide comprises a nucleotidesequence that has at least 85% identity to the polynucleotide of any oneof (a) to (c). Yet more preferably, the polynucleotide comprises anucleotide sequence that has at least 90% identity to the polynucleotideof any one of (a) to (c). More preferably, the polynucleotide comprisesa nucleotide sequence that has at least 95% identity to thepolynucleotide of any one of (a) to (c). Most preferably, thepolynucleotide comprises a nucleotide sequence that has at least 98%identity to the polynucleotide of any one of (a) to (c).

The polynucleotide described above preferably encodes a G-proteincoupled receptor (GPCR). The present invention also provides apolynucleotide probe or primer comprising at least 15 contiguousnucleotides of the polynucleotide described above.

The present invention yet further provides a vector comprising thepolynucleotide described above.

According to a further aspect of the present invention, there isprovided a host cell transformed or transfected with the vectordescribed above. Preferably, the host cell is a mammalian, insect,fungal, bacterial or yeast cell.

According to a further aspect of the present invention, there isprovided the transcribed RNA product of the polynucleotide describedabove. There is also provided an RNA molecule or a fragment thereofwhich is antisense in relation to the RNA product and is capable ofhybridising thereto.

There is yet further provided a ribozyme or zinc finger protein capableof binding to the polynucleotide described above.

According to yet a further aspect of the present invention, there isprovided a process for producing a polypeptide or fragment thereofcomprising culturing said host cell under conditions sufficient for theexpression of said polypeptide or fragment. Preferably, said polypeptideor fragment is expressed at the surface of said cell. The processpreferably further includes recovering the polypeptide or fragment fromthe culture.

There is also provided by the present invention a process for producingcells capable of expressing a polypeptide or fragment thereof comprisingtransforming or transfecting cells with the vector described above.

According to a further embodiment of the present invention, there areprovided cells produced by the process described above. There is alsoprovided a membrane preparation of said cells.

According to another aspect of the present invention, there is provideda polypeptide comprising:

-   -   (a) a polypeptide having the deduced amino acid sequence        translated from the polynucleotide sequence in SEQ ID NO: 1 and        variants, fragments, homologues, analogues and derivatives        thereof;    -   (b) a polypeptide of SEQ ID NO: 2 and variants, fragments,        homologues, analogues and derivatives thereof; or    -   (c) a polypeptide encoded by the cDNA of NCIMB 41066 and        variants, fragments, homologues, analogues and derivatives        thereof.

There is also provided by the present invention an antibody against thepolypeptide described above.

The present invention yet further provides a compound, which modulatesthe polypeptide described above. Preferably, the compound antagonises orselectively antagonises the polypeptide. Alternatively, the compoundagonises the polypeptide.

Also provided by the present invention is a pharmaceutical compositioncomprising the antibody or compound described above and one or morepharmaceutically acceptable carriers, diluents, adjuvants or excipients.

According to another aspect of the present invention, there is provideda method for identifying a compound, which binds to and modulates thepolypeptide described above comprising contacting said polypeptide witha candidate compound and determining whether modulation occurs.

Preferably, said method comprises:

-   -   (a) contacting a compound with cells expressing the polypeptide        described above on their cell surface, said polypeptide being        associated with a second component capable of providing a        detectable signal in response to the binding of a compound to        said polypeptide; said contacting being under conditions        sufficient to permit binding of compounds to the polypeptide;        and    -   (b) identifying a compound capable of polypeptide binding by        detecting the signal produced by said second component.

Alternatively, said method comprises:

-   -   (a) contacting (i) a detectable first component known to bind to        the polypeptide described above and (ii) a compound, with cells        expressing the above polypeptide on their cell surface, said        polypeptide being associated with a second component capable of        providing a detectable signal in response to the binding of a        compound to said polypeptide; said contacting being under        conditions sufficient to permit binding of compounds to the        polypeptide; and    -   (b) determining whether the first component binds to the        polypeptide by detecting the absence or otherwise of a signal        generated from the interaction of the first component with the        polypeptide.

The compound identified by any of the above methods preferably binds toand (i) antagonises or selectively antagonises the polypeptide describedabove, or (ii) agonises the polypeptide described above.

As GPCRs are involved in signal transduction, modulators (e.g. agonistsor antagonists) of the polypeptide of the present invention can find usein interfering in the signal transduction process.

Therefore, according to yet another embodiment of the present invention,there is provided the antibody, compound or composition described abovefor use as a pharmaceutical.

Such antibodies, compounds and compositions, which can modulate thepolypeptide of the present invention, can therefore find use in thetherapeutic areas which concern aspects of signal transduction.Therapeutically usefully areas include, but are not limited to, obesity,diabetes and metabolic disease, neurological disease,psychotherapeutics, urogenital disease, reproduction and sexualmedicine, inflammation, cancer, tissue repair, dermatology, skinpigmentation, photoaging, frailty, osteoporosis, cardiovascular disease,gastrointestinal disease, antiinfection, allergy and respiratorydisease, sensory organ disorders, sleep disorders and hairloss.

Accordingly, there is also provided the use of the compound describedabove in the manufacture of a medicament for the treatment of a patienthaving need to modulate the polypeptide described above. Preferably, thetreatment is for a patient having need to antagonise or selectivelyantagonise the polypeptide. Alternatively, the treatment is for apatient having need to agonise the polypeptide.

According to yet a further aspect of the invention, there is provided amethod for the treatment of a patient having need to modulate thepolypeptide described above comprising administering to the patient atherapeutically effective amount of the above-described compound.Preferably, said method is for the treatment of a patient having need toantagonise or selectively antagonise the polypeptide. Alternatively,said method is for the treatment of a patient having need to agonise thepolypeptide.

Preferably, said compound is a polypeptide and a therapeuticallyeffective amount of the compound is administered by providing to thepatient DNA encoding said compound and expressing said compound in vivo.

There is also provided by the present invention use of the antibodydescribed above in the manufacture of a medicament for the treatment ofa patient having need to modulate the polypeptide described above.Preferably, said method is for the treatment of a patient having need toantagonise or selectively antagonise the polypeptide. Alternatively,said method is for the treatment of a patient having need to agonise thepolypeptide.

Yet further provided by the present invention is a method for thetreatment of a patient having need to modulate the polypeptide describedabove, comprising administering to the patient a therapeuticallyeffective amount of the antibody described above. Preferably, saidmethod is for the treatment of a patient having need to antagonise orselectively antagonise the polypeptide. Alternatively, said method isfor the treatment of a patient having need to agonise the polypeptide.

The present invention also provides use of the compound described abovein the manufacture of a medicament for the treatment of obesity.

Yet further provided by the present invention is use of the antibodydescribed above in the manufacture of a medicament for the treatment ofobesity.

The present invention also provides a method for the treatment ofobesity in a patient comprising administering to the patient atherapeutically effective amount of the compound described above.

Yet further provided by the present invention is a method for thetreatment of obesity in a patient comprising administering to thepatient a therapeutically effective amount of the antibody describedabove.

According to yet a further aspect of the present invention, there areprovided cells genetically engineered ex vivo or in vivo to express,overexpress, underexpress or to exhibit targeted insertion or deletionof the polypeptide of the present invention.

PFI-002 Polypeptide

As explained above, the present invention relates to a novel GPCR—whichhas been internally designated PFI-002—and to a nucleotide sequenceencoding same. The present invention also relates to the use of thenovel nucleic acid and amino acid sequences in the diagnosis andtreatment of disease. The present invention also relates to the use ofthe novel nucleic acid and amino acid sequences to evaluate and/or toscreen for agents that can modulate the GPCR. The present inventionfurther relates to genetically engineered host cells that comprise orexpress the novel nucleic acid and amino acid sequences to evaluateand/or to screen for agents that can modulate the GPCR.

The PFI-002 polypeptide may be the same as the naturally occurringform—for this aspect, preferably the PFI-002 polypeptide is thenon-native amino acid sequence (i.e. it is not present in its naturalenvironment)—or is a variant, homologue, fragment or derivative thereof.In addition, or in the alternative, the PFI-002 polypeptide is isolatedPFI-002 polypeptide and/or purified PFI-002 polypeptide. The PFI-002polypeptide can be obtainable from or produced by any suitable source,whether natural or not, or it may be synthetic, semi-synthetic orrecombinant.

The PFI-002 coding sequence may be the same as the naturally occurringform—for this aspect, preferably the PFI-002 coding sequence is thenon-native nucleotide sequence (i.e. it is not present in its naturalenvironment)—or is a variant, homologue, fragment or derivative thereof.In addition, or in the alternative, the PFI-002 coding sequence is anisolated PFI-002 coding sequence and/or a purified PFI-002 codingsequence. The PFI-002 coding sequence can be obtainable from or producedby any suitable source, whether natural or not, or it may be synthetic,semi-synthetic or recombinant.

PFI-002 Polypeptide and Screening

The PFI-002 polypeptide and/or its coding sequence and/or a sequencecapable of hybridising thereto is/are useful for testing the selectivityof drug candidates between different GPCRs.

It has been demonstrated (herein) that PFI-002 is most closely similarto neurotensin receptors and that PFI-002 encodes a novel GPCR whoseligand is likely to be a peptide.

Drugs that modulate the novel PFI-002 receptor will therefore be likelyto modulate signal transduction processes. It is therefore likely thatmodulators of the PFI-002 receptor may be useful for the treatment ofmany different disorders associated with signal transduction that willmost likely include, but is not limited to, obesity, diabetes andmetabolic disease, neurological disease, psychotherapeutics, urogenitaldisease, reproduction and sexual medicine, inflammation, cancer, tissuerepair, dermatology, skin pigmentation, photoaging, frailty,osteoporosis, cardiovascular disease, gastrointestinal disease,antiinfection, allergy and respiratory disease, sensory organ disorders,sleep disorders and hairloss.

Thus, the PFI-002 polypeptide and/or its coding sequence and/or asequence capable of hybridising thereto may be useful for screening drugcandidates for the treatment of diseases associated with signaltransduction. In addition, it is believed that PFI-002 polypeptideand/or its coding sequence and/or a sequence capable of hybridisingthereto may be useful for screening drug candidates for the treatment ofdiseases such as those described above.

Either or both of the nucleotide sequences coding for the PFI-002polypeptide or the PFI-002 polypeptide itself may be used to screen foragents that can affect GPCR activity. In particular, the nucleotidesequence coding for the PFI-002 receptor itself may be used to screenfor agents that can antagonise GPCR activity. In addition, thenucleotide sequence coding for PFI-002 polypeptide or the PFI-002polypeptide itself may be used to screen for agents that selectivelyaffect GPCR activity, such as selectively antagonise the PFI-002receptor.

Polypeptide of the Present Invention

The term “polypeptide”—which is interchangeable with the term“protein”—includes single-chain polypeptide molecules as well asmultiple-polypeptide complexes where individual constituent polypeptidesare linked by covalent or non-covalent means.

Preferably, the polypeptide of the present invention is a single-chainpolypeptide.

Polypeptides of the present invention may be in a substantially isolatedform. It will be understood that the polypeptide may be mixed withcarriers or diluents which will not interfere with the intended purposeof the polypeptide and still be regarded as substantially isolated. Apolypeptide of the present invention may also be in a substantiallypurified form, in which case it will generally comprise the polypeptidein a preparation in which more than 90%, e.g. 95%, 98% or 99% of thepolypeptide in the preparation is a polypeptide of the presentinvention. Polypeptides of the present invention may be modified forexample by the addition of histidine residues to assist theirpurification.

Polypeptides of the present invention may be produced by synthetic means(e.g. as described by Geysen et al., 1996) or recombinantly, asdescribed below.

In a preferred embodiment, the amino acid sequence per se of the presentinvention does not cover the native PFI-002 receptor according to thepresent invention when it is in its natural environment and when it hasbeen expressed by its native nucleotide coding sequence which is also inits natural environment and when that nucleotide sequence is under thecontrol of its native promoter which is also in its natural environment.For ease of reference, we have called this preferred embodiment the“non-native amino acid sequence”.

The terms “variant”, “homologue”, “fragment”, “analogue” or “derivative”in relation to the amino acid sequence for the polypeptide of thepresent invention include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) amino acid from or to the sequence providing the resultantpolypeptide has GPCR activity, preferably being at least as biologicallyactive as the polypeptide shown in attached SEQ ID NO: 2. In particular,the term “homologue” covers homology with respect to structure and/orfunction. With respect to sequence homology, there is at least 70%,preferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least 95%homology to the sequence shown in SEQ ID NO: 2. Most preferably there isat least 98% homology to the sequence shown in SEQ ID NO: 2.

Typically, for the variant, homologue or fragment of the presentinvention, the types of amino acid substitutions that could be madeshould maintain the hydrophobicity/hydrophilicity of the amino acidsequence. Amino acid substitutions may be made, for example from 1, 2 or3 to 10, 20 or 30 substitutions provided that the modified sequenceretains the ability to act as a GPCR in accordance with the presentinvention. Amino acid substitutions may include the use of non-naturallyoccurring analogues.

The amino acid sequence of the present invention may be produced byexpression of a nucleotide sequence coding for same in a suitableexpression system.

In addition, or in the alternative, the protein itself could be producedusing chemical methods to synthesize a PFI-002 polypeptide, in whole orin part. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (e.g. Creighton (1983) ProteinsStructures and Molecular Principles, WH Freeman and Co., New York, N.Y.,USA). The composition of the synthetic peptides may be confirmed byamino acid analysis or sequencing (e.g. the Edman degradationprocedure).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge J Y et al Science Vol 269 1995 202-204) andautomated synthesis may be achieved, for example, using the ABI 431 APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer. Additionally, the amino acid sequence ofPFI-002, or any part thereof, may be altered during direct synthesisand/or combined using chemical methods with a sequence from othersubunits, or any part thereof, to produce a variant polypeptide.

In another embodiment of the invention, a PFI-002 natural, modified orrecombinant amino acid sequence may be ligated to a heterologoussequence to encode a fusion protein. For example, for screening oflibraries for compounds and peptide agonists and antagonists of PFI-002GPCR activity, it may be useful to encode a chimeric PFI-002 proteinexpressing a heterologous epitope that is recognised by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between a PFI-002 sequence and the heterologousprotein sequence, so that the PFI-002 may be cleaved and purified awayfrom the heterologous moiety.

PFI-002 may also be expressed as a recombinant protein with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilised metals (Porath J, Protein Expr Purif Vol 31992 p263-281), protein A domains that allow purification on immobilisedimmunoglobulin, and the domain utilised in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash., USA). The inclusionof a cleavable linker sequence such as Factor XA or enterokinase(Invitrogen, San Diego, Calif., USA) between the purification domain andPFI-002 is useful to facilitate purification.

A specific amino acid sequence of PFI-002 is shown in SEQ ID NO: 2.However, the present invention encompasses amino acid sequences encodingother GPCRs which would include amino acid sequences having at least 70%identity (preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95%, most preferably at least 98% identity) to that specificamino acid sequence.

Polypeptides of the present invention also include fragments of thepresent amino acid sequence and variants thereof. Suitable fragmentswill be at least 5, e.g. at least 10, 12, 15 or 20 amino acids in size.

Polypeptides of the present invention may also be modified to containone or more (e.g. at least 2, 3, 5, or 10) substitutions, deletions orinsertions, including conserved substitutions. These aspects arediscussed in a later section.

Nucleotide Sequence of the Present Invention

The term “nucleotide sequence” as used herein refers to anoligonucleotide sequence or polynucleotide sequence, and variants,homologues, fragments, analogues and derivatives thereof (such asportions thereof). The nucleotide sequence may be DNA or RNA which maybe of genomic or synthetic or recombinant origin which may bedouble-stranded or single-stranded whether representing the sense orantisense strand.

Preferably, the term “nucleotide sequence” means DNA.

More preferably, the term “nucleotide sequence” means DNA prepared byuse of recombinant DNA techniques (i.e. recombinant DNA).

In a preferred embodiment, the nucleotide sequence per se of the presentinvention does not cover the native nucleotide coding sequence accordingto the present invention in its natural environment when it is under thecontrol of its native promoter which is also in its natural environment.For ease of reference, we have called this preferred embodiment the“non-native nucleotide sequence”.

The nucleotide sequences of the present invention may include withinthem synthetic or modified nucleotides. A number of different types ofmodification to oligonucleotides are known in the art. These includemethylphosphonate and phosphorothioate backbones, addition of acridineor polylysine chains at the 3′ and/or 5′ ends of the molecule. For thepurposes of the present invention, it is to be understood that thenucleotide sequences described herein may be modified by any methodavailable in the art. Such modifications may be carried out in toenhance the in vivo activity or life span of nucleotide sequences of thepresent invention.

The present invention also encompasses nucleotide sequences that arecomplementary to the sequences presented herein, or any variant,homologue, analogue, fragment or derivative thereof. If the sequence iscomplementary to a fragment thereof then that sequence can be used aprobe to identify similar coding sequences in other organisms, etc.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences presented herein, or anyvariant, homologue, analogue, fragment or derivative thereof.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences that are complementary to thesequences presented herein, or any variant, homologue, analogue,fragment or derivative thereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hydridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hydridising understringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015Na₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of the present invention (including complementary sequences ofthose presented herein).

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridising to thenucleotide sequences presented herein under conditions of intermediateto maximal stringency.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present invention,or the complement thereof, under stringent conditions (e.g. 65° C. and0.1×SSC).

Exemplary nucleic acids can alternatively be characterised as thosenucleotide sequences which encode a PFI-002 protein and hybridise to theDNA sequence shown in SEQ ID NO: 1. Preferred are such sequencesencoding PFI-002 which hybridise under high-stringency conditions to thesequence shown in SEQ ID NO: 1 or the complement thereof.

Advantageously, the invention provides nucleic acid sequences which arecapable of hybridising, under stringent conditions, to a fragment of thesequence shown in the SEQ ID NO: 1 or the complement thereof.Preferably, the fragment is between 15 and 50 bases in length.Advantageously, it is about 25 bases in length.

The terms “variant”, “homologue”, “analogue”, “derivative” or “fragment”in relation to the nucleotide sequence coding for the preferredpolypeptide of the present invention include any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) nucleic acid from or to the sequence providing theresultant nucleotide sequence codes for or is capable of coding for anpolypeptide having PFI-002 receptor activity, preferably being at leastas biologically active as the polypeptide encoded by the sequence shownin SEQ ID NO: 1. In particular, the term “homologue” covers homologywith respect to structure and/or function providing the resultantnucleotide sequence codes for or is capable of coding for a polypeptidehaving activity as a PFI-002 GPCR. With respect to sequence homology,preferably there is at least 70%, preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95% homology to a nucleotidesequence coding for the amino acid sequence shown in SEQ ID NO: 2. Mostpreferably there is at least 98% homology to a nucleotide sequencecoding for the amino acid sequence shown in SEQ ID NO: 2. With respectto sequence homology, there is at least 70%, preferably at least 75%,more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 95% homology to thenucleotide sequence shown in SEQ ID NO: 1. Most preferably there is atleast 98% homology to the nucleotide sequence shown in SEQ ID NO: 1.

As indicated, the present invention relates to a DNA sequence(preferably a cDNA sequence) encoding PFI-002. In particular, thepresent invention relates to cDNA sequences encoding PFI-002.

The present invention also relates to DNA segments comprising the DNAsequence shown in SEQ ID NO: 1 or allelic variations thereof.

The present invention also relates to polypeptides produced byexpression in a host cell into which has been incorporated the foregoingDNA sequences or allelic variations thereof.

The present invention also relates provides DNA comprising the DNAsequence shown in SEQ ID NO: 1 or allelic variations thereof.

The present invention also relates to non-native DNA comprising the DNAsequence shown in SEQ ID NO: 1 or allelic variations thereof.

A highly preferred aspect of the present invention relates torecombinant DNA comprising the DNA sequence shown in SEQ ID NO: 1 orallelic variations thereof.

Polynucleotides of the present invention include nucleic acid sequencesencoding the polypeptides of the present invention. It will beappreciated that a range of different polynucleotides encode a givenamino acid sequence as a consequence of the degeneracy of the geneticcode.

By knowledge of the amino acid sequences set out herein it is possibleto devise partial and full-length nucleic acid sequences such as cDNAand/or genomic clones that encode the polypeptides of the presentinvention. For example, polynucleotides of the present invention may beobtained using degenerate polymerase chain reaction (PCR) which will useprimers designed to target sequences encoding the amino acid sequencespresented herein. The primers will typically contain multiple degeneratepositions. However, to minimise degeneracy, sequences will be chosenthat encode regions of the amino acid sequences presented hereincontaining amino acids such as methionine which are coded for by onlyone triplet. In addition, sequences will be chosen to take into accountcodon usage in the organism whose nucleic acid is used as the templateDNA for the PCR procedure. PCR will be used at stringency conditionslower than those used for cloning sequences with single sequence(non-degenerate) primers against known sequences.

Nucleic acid sequences obtained by PCR that encode polypeptide fragmentsof the present invention may then be used to obtain larger sequencesusing hybridisation library screening techniques. For example a PCRclone may be labelled with radioactive atoms and used to screen a cDNAor genomic library from other species, preferably other mammalianspecies. Hybridisation conditions will typically be conditions of mediumto high stringency (for example 0.03M sodium chloride and 0.03M sodiumcitrate at from about 50° C. to about 60° C.). Degenerate nucleic acidprobes encoding all or part of the amino acid sequence may also be usedto probe cDNA and/or genomic libraries from other species, preferablyother mammalian species. However, it is preferred to carry out PCRtechniques initially to obtain a single sequence for use in furtherscreening procedures.

In accordance with the present invention, polynucleotide sequences whichencode PFI-002, fragments of the polypeptide, fusion proteins orfunctional equivalents thereof, may be used to generate recombinant DNAmolecules that direct the expression of PFI-002 in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences which encode substantially the same or a functionallyequivalent amino acid sequence, may be used to clone and expressPFI-002. As will be understood by those of skill in the art, it may beadvantageous to produce PFI-002-encoding nucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particularprokaryotic or eukaryotic host (Murray E et al (1989) Nuc Acids Res17:477-508) can be selected, for example, to increase the rate ofPFI-002 expression or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, than transcriptsproduced from naturally occurring sequence.

Polynucleotide sequences of the present invention obtained using thetechniques described above may be used to obtain further homologoussequences and variants using the techniques described above. They mayalso be modified for use in expressing the polypeptides of the presentinvention in a variety of host cells systems, for example to optimisecodon preferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Altered PFI-002 polynucleotide sequences which may be used in accordancewith the invention include deletions, insertions or substitutions ofdifferent nucleotide residues resulting in a polynucleotide that encodesthe same or a functionally equivalent PFI-002. The protein may also havedeletions, insertions or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent PFI-002.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of PFI-002 is retained. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values includeleucine, isoleucine, valine, glycine, alanine, asparagine, glutamine,serine, threonine, phenylalanine, and tyrosine.

Included within the scope of the present invention are alleles ofPFI-002. As used herein, an “allele” or “allelic sequence” is analternative form of PFI-002. Alleles result from a mutation, i.e. achange in the nucleic acid sequence, and generally produce altered mRNAsor polypeptides whose structure or function may or may not be altered.Any given gene may have none, one or many allelic forms. Commonmutational changes which give rise to alleles are generally ascribed todeletions, additions or substitutions of amino acids. Each of thesetypes of changes may occur alone, or in combination with the others, oneor more times in a given sequence.

The nucleotide sequences of the present invention may be engineered inorder to alter a PFI-002 coding sequence for a variety of reasons,including but not limited to, alterations which modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art, e.g.site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns or to change codon preference.

Polynucleotides of the present invention may be used to produce aprimer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g. labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the present invention as used herein.

Polynucleotides or primers of the present invention may carry arevealing label. Suitable labels include radioisotopes such as ³²P or³⁵S, enzyme labels, or other protein labels such as biotin. Such labelsmay be added to polynucleotides or primers of the present invention andmay be detected using by techniques known in the art.

Polynucleotides such as a DNA polynucleotide and primers according tothe present invention may be produced recombinantly, synthetically, orby any means available to those of skill in the art. They may also becloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep-wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR cloning techniques. This will involvemaking a pair of primers (e.g. of about 15-30 nucleotides) to a regionof the nucleotide sequence which it is desired to clone, bringing theprimers into contact with mRNA or cDNA obtained from a eukaryotic orprokaryotic cell, performing a polymerase chain reaction underconditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixtureon an agarose gel) and recovering the amplified DNA. The primers may bedesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable cloning vector.

DNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences of the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule.

As mentioned earlier, the present invention also relates to nucleotidesequences that are capable of hybridising to all or part of the sequenceshown in SEQ ID NO: 1 or an allelic variation thereof. These nucleotidesequences may be used in antisense techniques to modify PFI-002expression. Alternatively, these sequences (or portions thereof) can beused as a probe, or for amplifying all or part of such sequence whenused as a PCR primer.

In addition to the recombinant DNA sequences, genomic sequences are alsoof utility in the context of drug discovery. It may be valuable toinhibit the mRNA transcription of a particular isoform rather than toinhibit its translated protein. This may be true with PFI-002, if thereare splice variants and wherein those different splice variants may betranscribed from different promoters.

Another utility of the invention is that the DNA sequences, once known,give the information needed to design assays to specifically detectisoforms or splice variants. Isoform-specific PCR primer pairs are butone example of an assay that depends completely on the knowledge of thespecific DNA sequence of the isoform or splice variant. Such an assayallows detection of mRNA for the isoform to access the tissuedistribution and biological relevance of each isoform to a particulardisease state. It also allows identification of cell lines that maynaturally express only one isoform—a discovery that might obviate theneed to express recombinant genes. If specific PFI-002 isoforms areshown to be associated with a particular disease state, the inventionwould be valuable in the design of diagnostic assays to detect thepresence of isoform mRNA.

An abnormal level of nucleotide sequences encoding a PFI-002 receptor ina biological sample may reflect a chromosomal aberration, such as anucleic acid deletion or mutation. Accordingly, nucleotide sequencesencoding a PFI-002 receptor provide the basis for probes which can beused diagnostically to detect chromosomal aberrations such as deletions,mutations or chromosomal translocations in the gene encoding PFI-002.PFI-002 gene expression may be altered in such disease states or theremay be a chromosomal aberration present in the region of the geneencoding PFI-002.

In an alternative embodiment of the invention, the coding sequence ofPFI-002 could be synthesized, in whole or in part, using chemicalmethods well known in the art (see Caruthers M H et al (1980) Nuc AcidsRes Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser225-232).

Naturally Occurring

As used herein “naturally occurring” refers to a PFI-002 with an aminoacid sequence found in nature.

Isolated/Purified

As used herein, the terms “isolated” and “purified” refer to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment and isolated or separated from at least one othercomponent with which they are naturally associated.

Biologically Active

As used herein “biologically active” refers to a PFI-002 according tothe present invention—such as a recombinant PFI-002—having a similarstructural function (but not necessarily to the same degree), and/orsimilar regulatory function (but not necessarily to the same degree),and/or similar biochemical function (but not necessarily to the samedegree) and/or immunological activity (but not necessarily to the samedegree) of the naturally occurring PFI-002. Specifically, a PFI-002 ofthe present invention has the ability to act as a GPCR, which is one ofthe characteristic activities of the PFI-002 polypeptide of the presentinvention.

Immunological Activity

As used herein, “immunological activity” is defined as the capability ofthe natural, recombinant or synthetic PFI-002 or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies.

Derivative

The term “derivative” as used herein in relation to the amino acidsequence includes chemical modification of a PFI-002. Illustrative ofsuch modifications would be replacement of hydrogen by an alkyl, acyl,or amino group.

Analogue

The term “analogue” as used herein in relation to the amino acidsequence (or the coding sequence thereof) includes chemical modificationof a PFI-002 or the coding sequence thereof. Illustrative of suchmodifications would be replacement of natural amino acid residues ornatural nucleotides with non-natural amino acid residues (e.g. D-aminoacids, beta-alanine, hydroxyproline) or non-natural nucleotides (e.g.inosine, demethyl-cytidine).

Deletion

As used herein a “deletion” is defined as a change in either nucleotideor amino acid sequence in which one or more nucleotides or amino acidresidues, respectively, are absent.

Insertion/Addiction

As used herein an “insertion” or “addition” is a change in a nucleotideor amino acid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring PFI-002.

Substitution

As used herein “substitution” results from the replacement of one ormore nucleotides or amino acids by different nucleotides or amino acids,respectively.

Homologue

The term “homologue” with respect to the nucleotide sequence of thepresent invention and the amino acid sequence of the present inventionmay be synonymous with allelic variations of the sequences.

In particular, the term “homology” as used herein may be equated withthe term “identity”. Here, sequence homology with respect to thenucleotide sequence of the present invention and the amino acid sequenceof the present invention can be determined by a simple “eyeball”comparison (i.e. a strict comparison) of any one or more of thesequences with another sequence to see if that other sequence has atleast 70% identity to the nucleotide and amino acid sequences. Relativesequence homology (i.e. sequence identity) can also be determined bycommercially available computer programs that can calculate percentage(%) homology between two or more sequences. Typical examples of suchcomputer programs are FASTA or BLAST.

Percentage (%) homology may be calculated over contiguous sequences,i.e. one sequence is aligned with the other sequence and each amino acidin one sequence directly compared with the corresponding amino acid inthe other sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Besffitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and 4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Besffit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software that can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Altschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor off-line and on-line searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However, for some applications it is preferred to use theGCG Bestfit program.

Although the final % homology can be measured in terms of identity, insome cases, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied (see user manual for further details). It is preferred to usethe public default values for the GCG package, or in the case of othersoftware, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

As indicated, for some applications, sequence homology (or identity) maybe determined using any suitable homology algorithm, using for exampledefault parameters. For a discussion of basic issues in similaritysearching of sequence databases, see Altschul et al. (1994) NatureGenetics 6:119-129. For some applications, the BLAST algorithm isemployed, with parameters set to default values. The BLAST algorithm isdescribed in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html.Advantageously, “substantial homology”, when assessed by BLAST, equatesto sequences which match with an EXPECT value of at least about e-7,preferably at least about e-9 and most preferably e-10 or lower. Thedefault threshold for EXPECT in BLAST searching is usually 10.

Should Gap Penalties be used when determining sequence identity, thenpreferably the following parameters are used: FOR BLAST GAP OPEN 5 GAPEXTENSION 2 FOR CLUSTAL DNA PROTEIN WORD SIZE 2 1 K triple GAP PENALTY10 10 GAP EXTENSION 0.1 0.1

Other computer program methods to determine identity and similaritybetween the two sequences include but are not limited to the GCG programpackage (Devereux et al., 1984 Nucleic Acids Research 12: 387) and FASTA(Atschul et al 1990 J Molec Biol 403-41 0).

Polypeptide Variants and Derivatives

The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence has PFI-002 activity, preferably having atleast the same activity as the polypeptide presented in SEQ ID NO: 2.

The sequences of the present invention may be modified for use in thepresent invention. Typically, modifications are made that maintain thePFI-002 activity of the sequence. Amino acid substitutions may be made,for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided thatthe modified sequence retains PFI-002 activity. Amino acid substitutionsmay include the use of non-naturally occurring analogues, for example toincrease blood plasma half-life of a therapeutically administeredpolypeptide.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R AROMATIC H F W Y

As indicated above, proteins of the invention are typically made byrecombinant means, for example as described herein, and/or by usingsynthetic means using techniques well known to the skilled person suchas solid phase synthesis. Variants and derivatives of such sequencesinclude fusion proteins, wherein the fusion proteins comprise at leastthe amino acid sequence of the present invention being linked (directlyor indirectly) to another amino acid sequence. These other amino acidsequences—which are sometimes referred to as fusion proteinpartners—will typically impart a favourable functionality—such as to aidextraction and purification of the amino acid sequence of the presentinvention. Examples of fusion protein partners includeglutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/ortranscriptional activation domains) and β-galactosidase. It may also beconvenient to include a proteolytic cleavage site between the fusionprotein partner and the protein sequence of the present invention so asto allow removal of the latter. Preferably the fusion protein partnerwill not hinder the function of the protein of the present invention.

Polynucleotide Variants and Derivatives

The terms “variant” or “derivative” in relation to the nucleotidesequence of the present invention include any substitution of, variationof, modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence codes for a polypeptide having PFI-002 activity,preferably having at least the same activity as the sequence presentedin SEQ ID NO: 2.

As indicated above, with respect to sequence homology, there is at least70%, preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95% homology to the nucleotide sequence shown in SEQ ID NO: 1.Most preferably there is at least 98% homology to the nucleotidesequence shown in SEQ ID NO: 1. Nucleotide homology comparisons may beconducted as described above. For some applications, a preferredsequence comparison program is the GCG Wisconsin Bestfit programdescribed above. The default scoring matrix has a match value of 10 foreach identical nucleotide and −9 for each mismatch. The default gapcreation penalty is −50 and the default gap extension penalty is −3 foreach nucleotide.

As used herein, the terms “variant”, “homologue”, “fragment” and“derivative” embrace allelic variations of the sequences.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hydridising to the nucleotide sequencespresented herein.

Hybridisation

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York, N.Y., USA) as well as the process of amplification ascarried out in PCR technologies as described in Dieffenbach C W and G SDveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., USA).

Hybridisation conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego, Calif., USA), and confer a defined“stringency” as explained below.

Stringency of hybridisation refers to conditions under which polynucleicacids hybrids are stable. Such conditions are evident to those ofordinary skill in the field. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (Tm) of thehybrid which decreases approximately 1 to 1.5° C. with every 1% decreasein sequence homology. In general, the stability of a hybrid is afunction of sodium ion concentration and temperature. Typically, thehybridisation reaction is performed under conditions of higherstringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permithybridisation of only those nucleic acid sequences that form stablehybrids in 1 M Na⁺ at 65-68° C.

Maximum stringency typically occurs at about Tm−5° C. (5° C. below theTm of the probe).

High stringency occurs at about 5° C. to 10° C. below the Tm of theprobe. High stringency conditions can be provided, for example, byhybridisation in an aqueous solution containing 6×SSC, 5× Denhardt's, 1%SDS (sodium dodecyl sulphate), 0.1 Na⁺ pyrophosphate and 0.1 mg/mldenatured salmon sperm DNA as non-specific competitor. Followinghybridisation, high stringency washing may be done in several steps,with a final wash (about 30 min) at the hybridisation temperature in0.2-0.1×SSC, 0.1% SDS.

Moderate, or intermediate, stringency typically occurs at about 10° C.to 20° C. below the Tm of the probe.

Low stringency typically occurs at about 20° C. to 25° C. below the Tmof the probe.

As will be understood by those of skill in the art, a maximum stringencyhybridisation can be used to identify or detect identical polynucleotidesequences while an intermediate (or low) stringency hybridisation can beused to identify or detect similar or related polynucleotide sequences.

Moderate stringency refers to conditions equivalent to hybridisation inthe above-described solution but at about 60-62° C. In that case thefinal wash is performed at the hybridisation temperature in 1×SSC, 0.1%SDS.

Low stringency refers to conditions equivalent to hybridisation in theabove-described solution at about 50-52° C. In that case, the final washis performed at the hybridisation temperature in 2×SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicatedusing a variety of buffers, e.g. formamide-based buffers, andtemperatures. Denhardt's solution and SSC are well known to those ofskill in the art as are other suitable hybridisation buffers (see, e.g.,Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, N.Y., USA or Ausubel, etal., eds. (1990) Current Protocols in Molecular Biology, John Wiley &Sons, Inc.). Optimal hybridisation conditions have to be determinedempirically, as the length and the GC content of the probe also play arole.

Polynucleotides of the invention capable of selectively hybridising tothe nucleotide sequences presented herein, or to their complement, willbe generally at least 70%, preferably at least 75%, more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 95%, most preferably at least 98% homologous tothe corresponding nucleotide sequences presented herein over a region ofat least 20, preferably at least 25 or 30, for instance at least 40, 60or 100 or more contiguous nucleotides.

The term “selectively hybridisable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide of theinvention is found to hybridize to the probe at a level significantlyabove background. The background hybridization may occur because ofother polynucleotides present, for example, in the cDNA or genomic DNAlibrary being screened. In this event, background implies a level ofsignal generated by interaction between the probe and a non-specific DNAmember of the library which is less than 10-fold, preferably less than100-fold as intense as the specific interaction observed with the targetDNA. The intensity of interaction may be measured, for example, byradiolabelling the probe, e.g. with ³²P.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to any one or more of the nucleotide sequences of thepresent invention under stringent conditions (e.g. 65° C. and 0.1×SSC{1×SSC=0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0}).

Where the polynucleotide of the present invention is double-stranded,both strands of the duplex, either individually or in combination, areencompassed by the present invention. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included within the scope of the presentinvention.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained, for example, by probing DNA libraries made froma range of individuals, for example individuals from differentpopulations. In addition, other viral/bacterial, or cellular homologuesparticularly cellular homologues found in mammalian cells (e.g. bovine,ovine, porcine, equine, rodent and primate cells), may be obtained andsuch homologues and fragments thereof in general will be capable ofselectively hybridising to the sequence shown in SEQ ID NO: 1. Suchsequences may be obtained by probing cDNA libraries made from, orgenomic DNA libraries derived from, other animal species, and probingsuch libraries with probes comprising all or part of the sequence shownin SEQ ID NO: 1 under conditions of medium to high stringency. Similarconsiderations apply to obtaining species homologues and allelicvariants of the polypeptide or nucleotide sequences of the invention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site-directedmutagenesis of characterised sequences. This may be useful where, forexample, silent codon changes are required to sequences to optimisecodon preferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Polynucleotides of the invention may be used to produce a primer, e.g. aPCR primer, a primer for an alternative amplification reaction, a probee.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, preferably at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to theinvention may be produced recombinantly, synthetically, or by any meansavailable to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR cloning techniques. This will involvemaking a pair of primers (e.g. of about 15 to 30 nucleotides) flanking aregion of the sequence which it is desired to clone, bringing theprimers into contact with mRNA or cDNA obtained from an animal or humancell, performing a polymerase chain reaction under conditions whichbring about amplification of the desired region, isolating the amplifiedfragment (e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector.

Regulatory Sequences

Preferably, the polynucleotide of the present invention is operablylinked to a regulatory sequence which is capable of providing for theexpression of the coding sequence, such as by the chosen host cell. Byway of example, the present invention covers a vector comprising thepolynucleotide of the present invention operably linked to such aregulatory sequence, i.e. the vector is an expression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site. Enhanced expression of the polynucleotideencoding the polypeptide of the present invention may also be achievedby the selection of heterologous regulatory regions, e.g. promoter,secretion leader and terminator regions, which serve to increaseexpression and, if desired, secretion levels of the protein of interestfrom the chosen expression host and/or to provide for the induciblecontrol of the expression of the polypeptide of the present invention.

Preferably, the nucleotide sequence of the present invention may beoperably linked to at least a promoter.

Aside from the promoter native to the gene encoding the polypeptide ofthe present invention, other promoters may be used to direct expressionof the polypeptide of the present invention. The promoter may beselected for its efficiency in directing the expression of thepolypeptide of the present invention in the desired expression host.

In another embodiment, a constitutive promoter may be selected to directthe expression of the desired polypeptide of the present invention. Suchan expression construct may provide additional advantages since itcircumvents the need to culture the expression hosts on a mediumcontaining an inducing substrate.

Examples of suitable promotors would be LTR, SV40 and CMV in mammaliansystems; E. coli lac or trp in bacterial systems; baculovirus polyhedronpromoter (polh) in insect systems and other promoters that are known tocontrol expression in eukaryotic and prokaryotic cells or their viruses.

Examples of strong constitutive and/or inducible promoters which arepreferred for use in fungal expression hosts are those which areobtainable from the fungal genes for xylanase (xlnA), phytase,ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpl),alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG—fromthe g/aA gene), acetamidase (amdS) and glyceraldehyde-3-phosphatedehydrogenase (gpd) promoters.

Examples of strong yeast promoters are those obtainable from the genesfor alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase andtriosephosphate isomerase.

Examples of strong bacterial promoters are the α-amylase and SP02promoters as well as promoters from extracellular protease genes.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct.

The promoter can additionally include features to ensure or to increaseexpression in a suitable host. For example, the features can beconserved regions such as a Pribnow Box or a TATA box. The promoter mayeven contain other sequences to affect (such as to maintain, enhance ordecrease) the levels of expression of the nucleotide sequence of thepresent invention. For example, suitable other sequences include theSh1-intron or an ADH intron. Other sequences include inducibleelements—such as temperature, chemical, light or stress inducibleelements. Also, suitable elements to enhance transcription ortranslation may be present. An example of the latter element is the TMV5′ signal sequence (see Sleat, Gene 217 [1987]217-225; and Dawson, PlantMol. Biol. 23 [1993]97).

The expression vector may also contain sequences which act on thepromoter to amplify expression. For example, the SV40, CMV, and polyomacis-acting elements (enhancer) and a selectable marker can provide aphenotypic trait for selection (e.g. dihydrofolate reductase or neomycinresistance for mammalian cells or amplicillin/tetracyclin resistance forE. coli). Selection of the appropriate vector containing the appropriatepromoter and selection marker is well within the level of those skilledin the art.

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes the nucleotide sequence according tothe present invention directly or indirectly attached to a promoter. Anexample of an indirect attachment is the provision of a suitable spacergroup such as an intron sequence, such as the Sh1-intron or the ADHintron, intermediate the promoter and the nucleotide sequence of thepresent invention. The same is true for the term “fused” in relation tothe present invention which includes direct or indirect attachment. Ineach case, the terms do not cover the natural combination of thenucleotide sequence coding for the protein ordinarily associated withthe wild-type gene promoter and when they are both in their naturalenvironment.

The construct may even contain or express a marker which allows for theselection of the genetic construct in, for example, mammalian, yeast,insect, fungal or bacterial cells. Various markers exist which may beused, such as, for example, those that provide for antibiotic resistanceto G418, hygromycin, bleomycin, kanamycin and gentamycin.

Preferably the construct of the present invention comprises at least thenucleotide sequence of the present invention operably linked to apromoter.

Vectors

The term “vector” includes expression vectors and transformation vectorsand shuttle vectors.

The term “expression vector” means a construct capable of in vivo or invitro expression.

The term “transformation vector” means a construct capable of beingtransferred from one entity to another entity—which may be of the samespecies or may be of a different species. If the construct is capable ofbeing transferred from one species to another—such as from a viralvector such as MMLV or FIV to a human or mammalian primary cell or cellline, then the transformation vector is sometimes referred to as a“shuttle vector”.

A large variety of expression systems may be used in different hosts.For example, episomal, chromosomal and virus-derived systems (e.g.vectors derived from bacterial plasmids, bacteriophage, papova virussuch as SV40, vaccinia virus, adenovirus, and retrovirus).

The DNA sequence can be inserted into the vector by a variety oftechniques. In general the DNA sequence is inserted into an appropriaterestriction endonuclease site by procedures known in the art and deemedto be within the scope of those skilled in the art. The DNA sequence inthe expression vector is linked operatively to appropriate controlsequences that direct mRNA synthesis (i.e the promoter).

The vectors of the present invention may be transformed into a suitablehost cell as described below to provide for expression of a polypeptideof the present invention. Thus, in a further aspect, the inventionprovides a process for preparing polypeptides according to the presentinvention which comprises cultivating a host cell transformed ortransfected with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the polypeptides, and recovering the expressed polypeptides.

The vectors may be, for example, plasmid, virus or bacteriophage (phage)vectors provided with an origin of replication, optionally a promoterfor the expression of the polynucleotide and optionally a regulator ofthe promoter.

The vectors of the present invention may contain one or more selectablemarker genes. The most suitable selection systems for industrialmicro-organisms are those formed by the group of selection markers whichdo not require a mutation in the host organism. Examples of fungalselection markers are the genes for acetamidase (amdS), ATP synthetase,subunit 9 (oliC), orotidine-5′-phosphate-decarboxylase (pvrA),phleomycin and benomyl resistance (benA). Examples of non-fungalselection markers are the bacterial G418 resistance gene (this may alsobe used in mammalian cells, yeast, but not in filamentous fungi), theampicillin resistance gene (E. coli), the neomycin resistance gene(mammalian cells) and the E. coli uidA gene, coding for β-glucuronidase(GUS).

Vectors may be used in vitro, for example for the production of RNA orused to transfect or transform a host cell.

Thus, polynucleotides of the present invention can be incorporated intoa recombinant vector (typically a replicable vector), for example acloning or expression vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Thus, in a further embodiment,the invention provides a method of making polynucleotides of the presentinvention by introducing a polynucleotide of the present invention intoa replicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells are described below in connection with expression vectors.

The present invention also relates to the use of genetically engineeredhost cells expressing a PFI-002 polypeptide or variant, homologue,fragment, analogue or derivative thereof in screening methods for theidentification of modulators (e.g. antagonists or agonists) of PFI-002.Such genetically engineered host cells could be used to screen peptidelibraries or organic molecules capable of modulating PFI-002 activity.Modulators (e.g. antagonists) of the PFI-002 polypeptide, such asantibodies, peptides or small organic molecules will provide the basisfor pharmaceutical compositions for the treatment of diseases associatedwith, for example, PFI-002. Such modulators (e.g. antagonists) can beadministered alone or in combination with other therapeutics for thetreatment of such diseases.

The present invention also relates to expression vectors and host cellscomprising polynucleotide sequences encoding PFI-002 or a variant,homologue, fragment, analogue or derivative thereof for the in vivo orin vitro production of PFI-002 protein or to screen for agents that canaffect PFI-002 expression or activity.

Tissue

The term “tissue” as used herein includes tissue per se and organ.

Host Cells

The term “host cell”—in relation to the present invention—includes anycell that could comprise the nucleotide sequence coding for therecombinant protein according to the present invention and/or productsobtained therefrom, wherein a promoter can allow expression of thenucleotide sequence according to the present invention when present inthe host cell.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a polynucleotide of the presentinvention. Preferably said polynucleotide is carried in a vector for thereplication and expression of said polynucleotide. The cells will bechosen to be compatible with the said vector and may, for example, beprokaryotic (for example, bacterial cells), or eukaryotic (i.e.mammalian, fungal, insect and yeast cells).

Introduction of polynucleotides into host cells can be effected bymethods as described in Sambrook, et al., eds. (1989) Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York,N.Y., USA. These methods include, but are not limited to, calciumphosphate transfection, DEAE-dextran-mediated transfection, cationiclipid-mediated transfection, electroporation, transvection,microinjection, transduction, scrape loading, and ballisticintroduction.

Examples of representative hosts include, bacterial cells (e.g E. coli,Steptomyces); fungal cells such as yeast cells and Aspergillus; insectcells such as Drosophila S2 and Spodoptera SF9 cells; animal cells suchas CHO, COS, HEK, HeLa, and 3T3 cells. The selection of the appropriatehost is deemed to be within the scope of those skilled in the art.

Depending on the nature of the polynucleotide encoding the polypeptideof the present invention, and/or the desirability for further processingof the expressed protein, eukaryotic hosts such as yeasts or other fungimay be preferred. In general, yeast cells are preferred over fungalcells because they are easier to manipulate. However, some proteins areeither poorly expressed or secreted from the yeast cell, or in somecases are not processed properly (e.g. hyperglycosylation in yeast). Inthese instances, a different fungal host organism should be selected.

Examples of suitable expression hosts within the scope of the presentinvention are fungi such as Aspergillus species (such as those describedin EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria suchas Escherichia species, Streptomyces species and Pseudomonas species;and yeasts such as Kluyveromyces species (such as those described inEP-A-0096430 and EP-A-0301670) and Saccharomyces species. By way ofexample, typical expression hosts may be selected from Aspergillusniger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori,Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae,Trichoderma reesei, Kluyveromyces lactis, Schizosaccharomyces pombe,Pichia pastoris and Saccharomyces cerevisiae.

The use of suitable host cells—such as mammalian, yeast, insect andfungal host cells—may provide for post-translational modifications (e.g.myristoylation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the presentinvention.

Organism

The term “organism” in relation to the present invention includes anyorganism, except man, that could comprise the nucleotide sequence codingfor the recombinant protein according to the present invention and/orproducts obtained therefrom, wherein a promoter can allow expression ofthe nucleotide sequence according to the present invention when presentin the organism. Examples of organisms may include a fungus, yeast orprotozoan.

The term “transgenic organism” in relation to the present inventionincludes any organism, except man, that comprises the nucleotidesequence coding for the protein according to the present inventionand/or products obtained therefrom, wherein the promoter can allowexpression of the nucleotide sequence according to the present inventionwithin the organism. Preferably the nucleotide sequence is incorporatedin the genome of the organism.

The term “transgenic organism” does not cover the native nucleotidecoding sequence according to the present invention in its naturalenvironment when it is under the control of its native promoter which isalso in its natural environment. In addition, the present invention doesnot cover the native protein according to the present invention when itis in its natural environment and when it has been expressed by itsnative nucleotide coding sequence which is also in its naturalenvironment and when that nucleotide sequence is under the control ofits native promoter which is also in its natural environment.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, the nucleotidesequence coding for the amino acid sequence according to the presentinvention, constructs according to the present invention (includingcombinations thereof), vectors according to the present invention,plasmids according to the present invention, cells according to thepresent invention, and tissues according to the present invention or theproducts thereof. The transformed cell or organism could prepareacceptable quantities of the desired compound which would be easilyretrievable from the cell or organism.

Transformation of Host Cells/Host Organisms

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. An example of a suitable prokaryotic host is E.coli. Teachings on the transformation of prokaryotic hosts are welldocumented in the art, for example see Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring HarborLaboratory Press, New York, N.Y., USA) and Ausubel et al. (CurrentProtocols in Molecular Biology (1995), John Wiley & Sons, Inc.).

In a preferred embodiment, the transformed host is a mammalian cell or,for example, an insect cell, wherein introduction of polynucleotidesinto said host cells can be effected by methods as described in, forexample, Sambrook et aL (Molecular Cloning: A Laboratory Manual, 2ndedition, 1989, Cold Spring Harbor Laboratory Press, New York, N.Y.,USA). These methods include, but are not limited to, calcium phosphatetransfection, DEAE-dextran-mediated transfection, cationiclipid-mediated transfection, electroporation, transvection,microinjection, transduction, scrape loading, and ballisticintroduction.

In another embodiment the transgenic organism can be a yeast. In thisregard, yeast have also been widely used as a vehicle for heterologousgene expression. The species Saccharomyces cerevisiae has a long historyof industrial use, including its use for heterologous gene expression.Expression of heterologous genes in Saccharomyces cerevisiae has beenreviewed by Goodey et al. (1987, Yeast Biotechnology, D R Berry et al,eds, pp 401-429, Allen and Unwin, London) and by King et al. (1989,Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds,pp 107-133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited forheterologous gene expression. First, it is non-pathogenic to humans andit is incapable of producing certain endotoxins. Second, it has a longhistory of safe use following centuries of commercial exploitation forvarious purposes. This has led to wide public acceptability. Third, theextensive commercial use and research devoted to the organism hasresulted in a wealth of knowledge about the genetics and physiology aswell as large-scale fermentation characteristics of Saccharomycescerevisiae.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe and E Kenny (“Yeast as a vehicle for the expression ofheterologous genes”, 1993, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrativevectors, which require recombination with the host genome for theirmaintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructsare prepared by inserting the nucleotide sequence of the presentinvention into a construct designed for expression in yeast. Severaltypes of constructs used for heterologous expression have beendeveloped. The constructs contain a promoter active in yeast fused tothe nucleotide sequence of the present invention, usually a promoter ofyeast origin, such as the GALL promoter, is used. Usually a signalsequence of yeast origin, such as the sequence encoding the SUC2 signalpeptide, is used. A terminator active in yeast ends the expressionsystem.

For the transformation of yeast several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al. (1978, Proceedings of the National Academy of Sciences of theUSA, 75: 1929); Beggs, J D (1978, Nature, London, 275:104); and Ito, Het al. (1983, J. Bacteriology 153:163-168).

The transformed yeast cells are selected using various selectivemarkers. Among the markers used for transformation are a number ofauxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibioticresistance markers such as aminoglycoside antibiotic markers, e.g. G418.

Thus, the present invention also provides a method of transforming ahost cell with a nucleotide sequence shown in SEQ ID NO: 1 or aderivative, homologue, variant, analogue or fragment thereof.

Host cells transformed with a PFI-002 nucleotide coding sequence may becultured under conditions suitable for the expression and recovery ofthe encoded protein (in cell membranes) from cell culture. The proteinproduced by a recombinant cell may be expressed on the cell surface,secreted or may be contained intracellularly depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing PFI-002 coding sequences willgenerally enable expression in the cell membrane. Other recombinantconstructions may join PFI-002 coding sequence to nucleotide sequenceencoding a polypeptide domain which will facilitate proteinpurification/identification (Kroll DJ et al. (1993) DNA Cell Biol Vol 12p441-53, see also above discussion of vectors containing fusionproteins).

Genetically Engineered or Genetically Modified

A cell, preferably an animal cell, that is “genetically modified” isheterozygous or homozygous for a modification that is introduced intothe cell, or into a progenitor cell, by genetic engineering. Thestandard methods of genetic engineering that are available forintroducing the modification include homologous recombination, viralvector gene trapping, irradiation, chemical mutagenesis, and thetransgenic expression of a nucleotide sequence encoding antisense RNAalone or in combination with catalytic ribozymes. Preferred methods forgenetic modification are homologous recombination and viral vector genetrapping which both modify an endogenous gene by inserting a foreignnucleic acid sequence into the gene locus. A nucleic acid sequence thatis foreign to the gene is an exogenous sequence that is non-naturallyoccurring in the gene. This insertion of foreign DNA can occur withinany region of the PFI-002 gene, e.g., in an enhancer, promoter,regulator region, non-coding region, coding region, intron, or exon. Themost preferred method of genetic engineering is homologousrecombination, in which the foreign nucleic acid sequence is inserted ina targeted manner either alone or in combination with a deletion of aportion of the endogenous gene sequence.

Functionally Disrupted

By a PFI-002 gene that is “functionally disrupted” is meant a PFI-002gene that is genetically modified such that the cellular activity of thePFI-002 polypeptide encoded by the disrupted gene is decreased in cellsthat normally express the wild-type version of the PFI-002 gene. Whenthe genetic modification effectively eliminates all wild-type copies ofthe PFI-002 gene in a cell (e.g., the genetically modified cell,preferably an animal cell, is homozygous for the PFI-002 gene disruptionor the only wild-type copy of PFI-002 gene originally present is nowdisrupted), then the genetic modification results in a reduction inPFI-002 polypeptide activity (i.e. reduced receptor expression) ascompared to an appropriate control cell that expresses the wild-typePFI-002 gene. This reduction in PFI-002 polypeptide activity (i.e.reduced receptor expression) results from either reduced PFI-002 geneexpression (i.e., PFI-002 mRNA levels are effectively reduced andproduce reduced levels of PFI-002 polypeptide) and/or because thedisrupted PFI-002 gene encodes a mutated polypeptide with reducedfunction or stability as compared to a wild-type PFI-002 polypeptide.Preferably, the activity (i.e. reduced receptor expression) of PFI-002polypeptide in the genetically modified cell is reduced to 50% or lessof wild-type levels, more preferably, to 25% or less, and, even morepreferably, to 10% or less of wild-type levels. Most preferably, thePFI-002 gene disruption results in a null mutation.

Genetically Modified Animal Cell

By a “genetically modified animal cell” containing a functionallydisrupted PFI-002 gene is meant an animal cell, including a human cell,created by genetic engineering to contain a functionally disruptedPFI-002 gene, as well as daughter cells that inherit the disruptedPFI-002 gene. These cells may be genetically modified in cultureaccording to any standard method known in the art. As an alternative togenetically modifying the cells in culture, non-human mammalian cellsmay also be isolated from a genetically modified, non-human mammal thatcontains a PFI-002 gene disruption. The animal cells of the inventionmay be obtained from primary cell or tissue preparations as well asculture-adapted, tumorigenic, or transformed cell lines. These cells andcell lines are derived, for example, from endothelial cells, epithelialcells, islets, neurons and other neural tissue-derived cells,mesothelial cells, osteocytes, lymphocytes, chondrocytes, hematopoieticcells, immune cells, cells of the major glands or organs (e.g., liver,lung, heart, stomach, pancreas, kidney, and skin), muscle cells(including cells from skeletal muscle, smooth muscle, and cardiacmuscle), exocrine or endocrine cells, fibroblasts, and embryonic andother totipotent or pluripotent stem cells (e.g., embryonic stem (ES)cells, ES-like cells, and embryonic germlne (EG) cells, and other stemcells, such as progenitor cells and tissue-derived stem cells). Thepreferred genetically modified cells are ES cells, more preferably,mouse or rat ES cells, and, most preferably, human ES cells.

A “homology region” used in a targeting vector for homologousrecombination with a PFI-002 gene is related (i.e., complementary) to aportion of the PFI-002 gene or a sequence flanking the PFI-002 gene to adegree sufficient to allow hybridization to occur between the homologyregion and the PFI-002 gene sequence under standard low stringencyconditions known in the art (e.g., as described in Current Protocols inHuman Genetics, unit 4.1, John Wiley & Sons, New York, N.Y., 2000).

By an “ES cell” or an “ES-like cell” is meant a pluripotent stem cellderived from an embryo, from a primordial germ cell, or from ateratocarcinoma, that is capable of indefinite self renewal as well asdifferentiation into cell types that are representative of all threeembryonic germ layers.

By “reduced” is meant a statistically significant decrease (i.e.,p<0.1).

The genetically modified animal cells, including human cells, of theinvention are heterozygous or homozygous for a modification thatfunctionally disrupts the PFI-002 gene. The animal cells may be derivedby genetically engineering cells in culture, or, in the case ofnon-human mammalian cells, the cells may be isolated from geneticallymodified, non-human mammals.

The PFI-002 gene locus is functionally disrupted by one of the severaltechniques for genetic modification known in the art, including chemicalmutagenesis (Rinchik, Trends in Genetics 7: 15-21, 1991, Russell,Environmental & Molecular Mutagenesis 23 (Suppl. 24) 23-29, 1994),irradiation (Russell, supra), transgenic expression of PFI-002 geneantisense RNA, either alone or in combination with a catalytic RNAribozyme sequence (Luyckx et al., Proc. Natl. Acad. Sci. 96:12174-79,1999; Sokol et al., Transgenic Research 5: 363-71, 1996; Efratet al., Proc. Natl. Acad. Sci. USA 91: 2051-55, 1994; Larsson et al.,Nucleic Acids Research 22: 2242-48, 1994) and, as further discussedbelow, the disruption of the PFI-002 gene by the insertion of a foreignnucleic acid sequence into the PFI-002 gene locus. Preferably, theforeign sequence is inserted by homologous recombination or by theinsertion of a viral vector. Most preferably, the method of PFI-002 genedisruption is homologous recombination and includes a deletion of aportion of the endogenous PFI-002 gene sequence.

The integration of the foreign sequence functionally disrupts thePFI-002 gene through one or more of the following mechanisms: byinterfering with the PFI-002 gene transcription or translation process(e.g., by interfering with promoter recognition, or by introducing atranscription termination site or a translational stop codon into thePFI-002 gene); or by distorting the PFI-002 gene coding sequence suchthat it no longer encodes a PFI-002 polypeptide with normal receptorfunction (e.g., by inserting a foreign coding sequence into the PFI-002gene coding sequence, by introducing a frameshift mutation or aminoacid(s) substitution, or, in the case of a double crossover event, bydeleting a portion of the PFI-002 gene coding sequence that is requiredfor expression of a functional receptor protein).

To insert a foreign sequence into a PFI-002 gene locus in the genome ofa cell, the foreign DNA sequence is introduced into the cell accordingto a standard method known in the art such as electroporation,calcium-phosphate precipitation, retroviral infection, microinjection,biolistics, liposome transfection, DEAE-dextran transfection, ortransferrinfection (see, e.g., Neumann et al., EMBO J. 1: 841-845, 1982;Potter et al., Proc. Natl. Acad. Sci USA 81: 7161-65, 1984; Chu et al.,Nucleic Acids Res. 15: 1311-26, 1987; Thomas and Capecchi, Cell 51:503-12, 1987; Baum et al., Biotechniques 17: 1058-62, 1994; Biewenga etal., J. Neuroscience Methods 71: 67-75, 1997; Zhang et al.,Biotechniques 15: 868-72, 1993; Ray and Gage, Biotechniques 13: 598-603,1992; Lo, Mol. Cell. Biol. 3: 1803-14, 1983; Nickoloff et al., Mol.Biotech. 10: 93-101, 1998; Linney et al., Dev. Biol. (Orlando) 213:207-16, 1999; Zimmer and Gruss, Nature 338: 150-153, 1989; and Robertsonet al., Nature 323: 445-48, 1986). The preferred method for introducingforeign DNA into a cell is electroporation.

Homologous Recombination

The method of homologous recombination targets the PFI-002 gene fordisruption by introducing a PFI-002 gene targeting vector into a cellcontaining a PFI-002 gene. The ability of the vector to target thePFI-002 gene for disruption stems from using a nucleotide sequence inthe vector that is homologous to the PFI-002 gene. This homology regionfacilitates hybridization between the vector and the endogenous sequenceof the PFI-002 gene. Upon hybridization, the probability of a crossoverevent between the targeting vector and genomic sequences greatlyincreases. This crossover event results in the integration of the vectorsequence into the PFI-002 gene locus and the functional disruption ofthe PFI-002 gene.

General principles regarding the construction of vectors used fortargeting are reviewed in Bradley et al. (Biotechnol. 10: 534, 1992).Two different exemplary types of vector can be used to insert DNA byhomologous recombination: an insertion vector or a replacement vector.An insertion vector is circular DNA which contains a region of PFI-002gene homology with a double stranded break. Following hybridizationbetween the homology region and the endogenous PFI-002 gene, a singlecrossover event at the double stranded break results in the insertion ofthe entire vector sequence into the endogenous gene at the site ofcrossover.

The more preferred vector to use for homologous recombination is areplacement vector, which is colinear rather than circular. Replacementvector integration into the PFI-002 gene requires a double crossoverevent, i.e. crossing over at two sites of hybridization between thetargeting vector and the PFI-002 gene. This double crossover eventresults in the integration of vector sequence that is sandwiched betweenthe two sites of crossover into the PFI-002 gene and the deletion of thecorresponding endogenous PFI-002 gene sequence that originally spannedbetween the two sites of crossover (see, e.g., Thomas and Capecchi etal., Cell 51: 503-12, 1987; Mansour et al., Nature 336: 348-52, 1988;Mansour et al., Proc. Natl. Acad. Sci. USA 87: 7688-7692, 1990; andMansour, GATA 7: 219-227, 1990).

A region of homology in a targeting vector is generally at least 100nucleotides in length. Most preferably, the homology region is at least1-5 kilobases (Kb) in length. Although there is no demonstrated minimumlength or minimum degree of relatedness required for a homology region,targeting efficiency for homologous recombination generally correspondswith the length and the degree of relatedness between the targetingvector and the PFI-002 gene locus. In the case where a replacementvector is used, and a portion of the endogenous PFI-002 gene is deletedupon homologous recombination, an additional consideration is the sizeof the deleted portion of the endogenous PFI-002 gene. If this portionof the endogenous PFI-002 gene is greater than 1 Kb in length, then atargeting cassette with regions of homology that are longer than 1 Kb isrecommended to enhance the efficiency of recombination. Further guidanceregarding the selection and use of sequences effective for homologousrecombination is described in the literature (see, e.g., Deng andCapecchi, Mol. Cell. Biol. 12: 3365-3371, 1992; Bollag et al., Annu.Rev. Genet. 23: 199-225, 1989; and Waldman and Liskay, Mol. Cell. Biol.8: 5350-5357,1988).

A wide variety of cloning vectors may be used as vector backbones in theconstruction of PFI-002 gene targeting vectors, includingpBluescript-related plasmids (e.g., Bluescript KS+11), pQE70, pQE60,pQE-9, pBS, pD10, phagescript, phiPFI-002174, pBK Phagemid, pNH8A,pNH16a, pNH18Z, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5PWLNEO, pSV2CAT, pPFI-002T1, pSG (Stratagene), pSVK3, PBPV, PMSG, andpSVL, pBR322 and pBR322-based vectors, pBM9, pBR325, pKH47, pBR328,pHC79, phage Charon 28, pKB11, pKSV-10, pK19 related plasmids, pUCplasmids, and the pGEM series of plasmids. These vectors are availablefrom a variety of commercial sources (e.g., Boehringer MannheimBiochemicals, Indianapolis, Ind.; Qiagen, Valencia, Calif.; Stratagene,La Jolla, Calif.; Promega, Madison, Wis.; and New England Biolabs,Beverly, Mass.). However, any other vectors, e.g. plasmids, viruses, orparts thereof, may be used as long as they are replicable and viable inthe desired host. The vector may also comprise sequences which enable itto replicate in the host whose genome is to be modified. The use of sucha vector can expand the interaction period during which recombinationcan occur, increasing the efficiency of targeting (see MolecularBiology, ed. Ausubel et al, Unit 9.16, FIG. 9.16.1).

The specific host employed for propagating the targeting vectorsdescribed above is not critical. Examples include E. coli K12 RR1(Bolivar et al., Gene 2: 95, 1977), E. coli K12 HB101 (ATCC No. 33694),E. coli MM21 (ATCC No. 336780), E. coli DH1 (ATCC No. 33849), E. colistrain DH5α, and E. coli STBL2. Alternatively, hosts such as C.cerevisiae can be used. The above-mentioned hosts are availablecommercially (e.g., Stratagene, La Jolla, Calif.; and Life Technologies,Rockville, Md.).

To create the targeting vector, a PFI-002 gene targeting construct isadded to an above-described vector backbone. The PFI-002 gene targetingconstructs described above have at least one PFI-002 gene homologyregion. To make the PFI-002 gene homology regions, a PFI-002gene-related sequence is used as a basis for producing polymerase chainreaction (PCR) primers. These primers are used to amplify the desiredregion of the PFI-002 sequence by high fidelity PCR amplification(Mattila et al., Nucleic Acids Res. 19: 4967, 1991; Eckert and Kunkel 1:17, 1991; and U.S. Pat. No. 4,683,202). The genomic sequence is obtainedfrom a genomic clone library or from a preparation of genomic DNA,preferably from the animal species that is to be targeted for PFI-002gene disruption.

Preferably, the targeting constructs described above also include anexogenous nucleotide sequence encoding a positive marker protein. Thestable expression of a positive marker after vector integration confersan identifiable characteristic on the cell without compromising cellviability. Therefore, in the case of a replacement vector, the markergene is positioned between two flanking homology regions so that itintegrates into the PFI-002 gene following the double crossover event.

It is preferred that the positive marker protein is a selectableprotein; the stable expression of such a protein in a cell confers aselectable phenotypic characteristic, i.e., the characteristic enhancesthe survival of the cell under otherwise lethal conditions. Thus, byimposing the selectable condition, one can isolate cells that stablyexpress the positive selectable marker from other cells that have notsuccessfully integrated the vector sequence on the basis of viability.Examples of positive selectable marker proteins (and their agents ofselection) include Neo (G418 or kanomycin), Hyg (hygromycin), HisD(histidinol), Gpt (xanthine), Ble (bleomycin), and Hprt (hypoxanthine)(see, e.g., Capecchi and Thomas, U.S. Pat. No. 5,464,764, and Capecchi,Science 244: 1288-92, 1989). Other positive markers that may also beused as an alternative to a selectable marker include reporter proteinssuch as β-galactosidase, firefly luciferase, or green fluorescentprotein (see, e.g., Current Protocols in Cytometry, Unit 9.5, andCurrent Protocols in Molecular Biology, Unit 9.6, John Wiley & Sons, NewYork, N.Y., 2000).

The above-described positive selection scheme does not distinguishbetween cells that have integrated the vector by targeted homologousrecombination at the PFI-002 gene locus versus random, non-homologousintegration of vector sequence into any chromosomal position. Therefore,when using a replacement vector for homologous recombination, it is alsopreferred to include a nucleotide sequence encoding a negativeselectable marker protein. Expression of a negative selectable markercauses a cell expressing the marker to lose viability when exposed to acertain agent (i.e., the marker protein becomes lethal to the cell undercertain selectable conditions). Examples of negative selectable markers(and their agents of lethality) include herpes simplex virus thymidinekinase (gancyclovir or1,2-deoxy-2-fluoro-α-d-arabinofuransyl-5-iodouracil), Hprt(6-thioguanine or 6-thioxanthine), and diphtheria toxin, ricin toxin,and cytosine deaminase (5-fluorocytosine).

The nucleotide sequence encoding the negative selectable marker ispositioned outside of the two homology regions of the replacementvector. Given this positioning, cells will only integrate and stablyexpress the negative selectable marker if integration occurs by random,non-homologous recombination; homologous recombination between thePFI-002 gene and the two regions of homology in the targeting constructexcludes the sequence encoding the negative selectable marker fromintegration. Thus, by imposing the negative condition, cells that haveintegrated the targeting vector by random, non-homologous recombinationlose viability.

The above-described combination of positive and negative selectablemarkers is preferred because a series of positive and negative selectionsteps can be designed to more efficiently select only those cells thathave undergone vector integration by homologous recombination, and,therefore, have a potentially disrupted PFI-002 gene. Further examplesof positive-negative selection schemes, selectable markers, andtargeting constructs are described, for example, in U.S. Pat. No.5,464,764, WO 94/06908, and Valancius and Smithies, Mol. Cell. Biol. 11:1402,1991.

In order for a marker protein to be stably expressed upon vectorintegration, the targeting vector may be designed so that the markercoding sequence is operably linked to the endogenous PFI-002 genepromoter upon vector integration. Expression of the marker is thendriven by the PFI-002 gene promoter in cells that normally expressPFI-002 gene. Alternatively, each marker in the targeting construct ofthe vector may contain its own promoter that drives expressionindependent of the PFI-002 gene promoter. This latter scheme has theadvantage of allowing for expression of markers in cells that do nottypically express the PFI-002 gene (Smith and Berg, Cold Spring HarborSymp. Quant. Biol. 49: 171, 1984; Sedivy and Sharp, Proc. Natl. Acad.Sci. (USA) 86: 227: 1989; Thomas and Capecchi, Cell 51: 503, 1987).

Exogenous promoters that can be used to drive marker gene expressioninclude cell-specific or stage-specific promoters, constitutivepromoters, and inducible or regulatable promoters. Non-limiting examplesof these promoters include the herpes simplex thymidine kinase promoter,cytomegalovirus (CMV) promoter/enhancer, SV40 promoters, PGK promoter,PMC1-neo, metallothionein promoter, adenovirus late promoter, vacciniavirus 7.5K promoter, avian beta globin promoter, histone promoters(e.g., mouse histone H3-614), beta actin promoter, neuron-specificenolase, muscle actin promoter, and the cauliflower mosaic virus 35Spromoter (see generally, Sambrook et al., Molecular Cloning, Vols.I-III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 2000; Stratagene, La Jolla, Calif.).

To confirm whether cells have integrated the vector sequence into thetargeted PFI-002 gene locus, primers or genomic probes that are specificfor the desired vector integration event can be used in combination withPCR or Southern blot analysis to identify the presence of the desiredvector integration into the PFI-002 gene locus (Erlich et al., Science252: 1643-51, 1991; Zimmer and Gruss, Nature 338: 150, 1989; Mouellic etal., Proc. Natl. Acad. Sci. (USA) 87: 4712, 1990; and Shesely et al.,Proc. Natl. Acad. Sci. (USA) 88: 4294, 1991).

Gene Trapping

Another method available for inserting a foreign nucleic acid sequenceinto the PFI-002 gene locus to functionally disrupt the PFI-002 gene isgene trapping. This method takes advantage of the cellular machinerypresent in all mammalian cells that splices exons into mRNA to insert agene trap vector coding sequence into a gene in a random fashion. Onceinserted, the gene trap vector creates a mutation that may functionallydisrupt the trapped PFI-002 gene. In contrast to homologousrecombination, this system for mutagenesis creates largely randommutations. Thus, to obtain a genetically modified cell that contains afunctionally dirsupted PFI-002 gene, cells containing this particularmutation must be identified and selected from a pool of cells thatcontain random mutations in a variety of genes.

Gene trapping systems and vectors have been described for use ingenetically modifying murine cells and other cell types (see, e.g.,Allen et al., Nature 333: 852-55, 1988; Bellen et al., Genes Dev. 3:1288-1300, 1989; Bier et al., Genes Dev. 3: 1273-1287, 1989; Bonnerot etal., J. Virol. 66: 4982-91, 1992; Brenner et al., Proc. Nat. Acad. Sci.USA 86: 5517-21, 1989; Chang et al., Virology 193: 737-47, 1993;Friedrich and Soriano, Methods Enzymol. 225: 681-701, 1993; Friedrichand Soriano, Genes Dev. 5: 1513-23, 1991; Goff, Methods Enzymol. 152:469-81, 1987; Gossler et al., Science 244: 463-65, 1989; Hope, Develop.113: 399-408, 1991; Kerr et al., Cold Spring Harb. Symp. Quant. Biol. 2:767-776, 1989; Reddy et al., J. Virol. 65: 1507-1515,1991; Reddy et al.,Proc. Natl. Acad. Sci. U.S.A. 89: 6721-25,1992; Skarnes et al., GenesDev. 6: 903-918, 1992; von Melchner and Ruley, J. Virol. 63: 3227-3233,1989; and Yoshida et al., Transgen. Res. 4: 277-87, 1995).

Promoter trap (5′ trap) vectors contain, in 5′ to 3′ order, a spliceacceptor sequence followed by an exon, which is typically characterizedby a translation initiation codon and open reading frame (ORF) and/or aninternal ribosome entry site. In general, these promoter trap vectors donot contain promoters or operably linked splice donor sequences.Consequently, after integration into the cellular genome of the hostcell, the promoter trap vector sequence intercepts the normal splicingof the upstream gene and acts as a terminal exon. Expression of thevector coding sequence is dependent upon the vector integrating into anintron of the disrupted gene in the proper reading frame. In such acase, the cellular splicing machinery splices exons from the trappedgene upstream of the vector coding sequence (Zambrowicz et al., WO99/50426).

An alternative method for producing an effect similar to theabove-described promoter trap vector is a vector that incorporates anested set of stop codons present in, or otherwise engineered into, theregion between the splice acceptor of the promoter trap vector and thetranslation initiation codon or polyadenylation sequence. The codingsequence can also be engineered to contain an independent ribosome entrysite (IRES) so that the coding sequence will be expressed in a mannerlargely independent of the site of integration within the host cellgenome. Typically, but not necessarily, an IRES is used in conjunctionwith a nested set of stop codons.

Another type of gene trapping scheme uses a 3′ gene trap vector. Thistype of vector contains, in operative combination, a promoter region,which mediates expression of an adjoining coding sequence, the codingsequence, and a splice donor sequence that defines the 3′ end of thecoding sequence exon. After integration into a host cell genome, thetranscript expressed by the vector promoter is spliced to a spliceacceptor sequence from the trapped gene that is located downstream ofthe integrated gene trap vector sequence. Thus, the integration of thevector results in the expression of a fusion transcript comprising thecoding sequence of the 3′ gene trap cassette and any downstream cellularexons, including the terminal exon and its polyadenylation signal. Whensuch vectors integrate into a gene, the cellular splicing machinerysplices the vector coding sequence upstream of the 3′ exons of thetrapped gene. One advantage of such vectors is that the expression ofthe 3′ gene trap vectors is driven by a promoter within the gene trapcassette and does not require integration into a gene that is normallyexpressed in the host cell (Zambrowicz et al., WO 99/50426). Examples oftranscriptional promoters and enhancers that may be incorporated intothe 3′ gene trap vector include those discussed above with respect totargeting vectors.

The viral vector backbone used as the structural component for thepromoter or 3′ gene trap vector may be selected from a wide range ofvectors that can be inserted into the genome of a target cell. Suitablebackbone vectors include, but are not limited to, herpes simplex virusvectors, adenovirus vectors, adeno-associated virus vectors, retroviralvectors, lentiviral vectors, pseudorabies virus, alpha-herpes virusvectors, and the like. A thorough review of viral vectors, inparticular, viral vectors suitable for modifying non-replicating cellsand how to use such vectors in conjunction with the expression of anexogenous polynucleotide sequence, can be found in Viral Vectors: GeneTherapy and Neuroscience Applications, Eds. Caplitt and Loewy, AcademicPress, San Diego, 1995.

Preferably, retroviral vectors are used for gene trapping. These vectorscan be used in conjunction with retroviral packaging cell lines such asthose described in U.S. Pat. No. 5,449,614. Where non-murine mammaliancells are used as target cells for genetic modification, amphotropic orpantropic packaging cell lines can be used to package suitable vectors(Ory et al., Proc. Natl. Acad. Sci., USA 93: 11400-11406, 1996).Representative retroviral vectors that can be adapted to create thepresently described 3′ gene trap vectors are described, for example, inU.S. Pat. No. 5,521,076.

The gene trapping vectors may contain one or more of the positive markergenes discussed above with respect to targeting vectors used forhomologous recombination. Similar to their use in targeting vectors,these positive markers are used in gene trapping vectors to identify andselect cells that have integrated the vector into the cell genome. Themarker gene may be engineered to contain an independent ribosome entrysite (IRES) so that the marker will be expressed in a manner largelyindependent of the location in which the vector has integrated into thetarget cell genome.

Given that gene trap vectors will integrate into the genome of infectedhost cells in a fairly random manner, a genetically modified cell havinga disrupted PFI-002 gene must be identified from a population of cellsthat have undergone random vector integration. Preferably, the geneticmodifications in the population of cells are of sufficient randomnessand frequency such that the population represents mutations inessentially every gene found in the cell's genome, making it likely thata cell with a disrupted PFI-002 gene will be identified from thepopulation (see Zambrowicz et al., WO 99/50426; Sands et al., WO98/14614).

Individual mutant cell lines containing a disrupted PFI-002 gene areidentified in a population of mutated cells using, for example, reversetranscription and PCR (RT-PCR) to identify a mutation in a PFI-002 genesequence. This process can be streamlined by pooling clones. Forexample, to find an individual clone containing a disrupted PFI-002gene, RT-PCR is performed using one primer anchored in the gene trapvector and the other primer located in the PFI-002 gene sequence. Apositive RT-PCR result indicates that the vector sequence is encoded inthe PFI-002 gene transcript, indicating that PFI-002 gene has beendisrupted by a gene trap integration event (see, e.g., Sands et al., WO98/14614).

Temporal, Spatial, and Inducible Gene Disruptions

A functional disruption of the endogenous PFI-002 gene can occur atspecific developmental or cell cycle stages (temporal disruption) or inspecific cell types (spatial disruption). The PFI-002 gene disruptioncan also be inducible when certain conditions are present. A recombinaseexcision system, such as a Cre-Lox system, may be used to activate orinactivate the PFI-002 gene at a specific developmental stage, in aparticular tissue or cell type, or under particular environmentalconditions. Generally, methods utilizing Cre-Lox technology are carriedout as described by Torres and Kuhn, Laboratory Protocols forConditional Gene Targeting, Oxford University Press, 1997. Methodologysimilar to that described for the Cre-Lox system can also be employedutilizing the FLP-FRT system. Further guidance regarding the use ofrecombinase excision systems for conditionally disrupting genes byhomologous recombination or viral insertion is provided, for example, inU.S. Pat. No. 5,626,159, U.S. Pat. No. 5,527,695, U.S. Pat. No.5,434,066, WO 98/29533, Orban et al., Proc. Nat. Acad. Sci. USA 89:6861-65, 1992; O'Gorman et al., Science 251: 1351-55, 1991; Sauer etal., Nucleic Acids Research 17: 147-61, 1989; Barinaga, Science 265:26-28, 1994; and Akagi et al., Nucleic Acids Res. 25: 1766-73, 1997.More than one recombinase system can be used to genetically modify ananimal cell.

When using homologous recombination to disrupt the PFI-002 gene in atemporal, spatial, or inducible fashion, using a recombinase system suchas the Cre-Lox system, a portion of the PFI-002 gene coding region isreplaced by a targeting construct comprising the PFI-002 gene codingregion flanked by loxP sites. Animal cells carrying this geneticmodification contain a functional, loxP-flanked PFI-002 gene. Thetemporal, spatial, or inducible aspect of the PFI-002 gene disruption iscaused by the expression pattern of an additional transgene, a Crerecombinase transgene, that is expressed in the animal cell under thecontrol of the desired spatially-regulated, temporally-regulated, orinducible promoter, respectively. A Cre recombinase targets the loxPsites for recombination. Therefore, when Cre expression is activated,the LoxP sites undergo recombination to excise the sandwiched PFI-002gene coding sequence, resulting in a functional disruption of thePFI-002 gene (Rajewski et al., J. Clin. Invest. 98: 600-03, 1996;St.-Onge et al., Nucleic Acids Res. 24: 3875-77, 1996; Agah et al., J.Clin. Invest. 100: 169-79, 1997; Brocard et al., Proc. Natl. Acad. Sci.USA 94: 14559-63, 1997; Feil et al., Proc. Natl. Acad. Sci. USA 93:10887-90, 1996; and Kuhn et al., Science 269: 1427-29, 1995).

A cell containing both a Cre recombinase transgene and loxP-flankedPFI-002 gene can be generated through standard transgenic techniques.Further guidance regarding the use of recombinase systems specificpromoters to temporally, spatially, or conditionally disrupt the PFI-002gene is found, for example, in Sauer, Meth. Enz. 225: 890-900, 1993, Guet al., Science 265: 103-06, 1994, Araki et al., J. Biochem. 122:977-82, 1997, Dymecki, Proc. Natl. Acad. Sci. 93: 6191-96, 1996, andMeyers et al., Nature Genetics 18: 136-41, 1998.

An inducible disruption of the PFI-002 gene can also be achieved byusing a tetracycline responsive binary system (Gossen and Bujard, Proc.Natl. Acad. Sci. USA 89: 5547-51, 1992). This system involvesgenetically modifying a cell to introduce a Tet promoter into theendogenous PFI-002 gene regulatory element and a transgene expressing atetracycline-controllable repressor (TetR). In such a cell, theadministration of tetracycline activates the TetR which, in turn,inhibits PFI-002 gene expression and, therefore, functionally disruptsthe PFI-002 gene (St.-Onge et al., Nucleic Acids Res. 24: 3875-77, 1996,U.S. Pat. No. 5,922,927).

The above-described systems for temporal, spatial, and inducibledisruptions of the PFI-002 gene can also be adopted when using genetrapping as the method of genetic modification, for example, asdescribed, for example, in WO 98/29533.

Creating Genetically Modified Animal Cells

The above-described methods for genetic modification can be used tofunctionally disrupt a PFI-002 gene in virtually any type of somatic orstem cell derived from an animal. Genetically modified animal cells ofthe invention include, but are not limited to, mammalian cells,including human cells, and avian cells. These cells may be derived fromgenetically engineering any animal cell line, such as culture-adapted,tumorigenic, or transformed cell lines, or they may be isolated from agenetically modified, non-human mammal carrying the desired PFI-002genetic modification.

The cells may be heterozygous or homozygous for the disrupted PFI-002gene. To obtain cells that are homozygous for the PFI-002 genedisruption (PFI-002−/−), direct, sequential targeting of both allelescan be performed. This process can be facilitated by recycling apositive selectable marker. According to this scheme the nucleotidesequence encoding the positive selectable marker is removed followingthe disruption of one allele using the Cre-Lox P system. Thus, the samevector can be used in a subsequent round of targeting to disrupt thesecond PFI-002 gene allele (Abuin and Bradley, Mol. Cell. Biol. 16:1851-56, 1996; Sedivy et al., T.I.G. 15: 88-90, 1999; Cruz et al., Proc.Natl. Acad. Sci. (USA) 88: 7170-74, 1991; Mortensen et al., Proc. Natl.Acad. Sci. (USA) 88: 7036-40, 1991; te Riele et al., Nature (London)348: 649-651, 1990).

An alternative strategy for obtaining ES cells that are PFI-002−/− isthe homogenotization of cells from a population of cells that isheterozygous for the PFI-002 gene disruption (PFI-002±). The method usesa scheme in which PFI-002± targeted clones that express a selectabledrug resistance marker are selected against a very high drugconcentration; this selection favours cells that express two copies ofthe sequence encoding the drug resistance marker and are, therefore,homozygous for the PFI-002 gene disruption (Mortensen et al., Mol. Cell.Biol. 12: 2391-95, 1992).

Following the genetic modification of the desired cell or cell line, thePFI-002 gene locus can be confirmed as the site of modification by PCRanalysis according to standard PCR or Southern blotting methods known inthe art (see, e.g., U.S. Pat. No. 4,683,202; and Erlich et al., Science252: 1643, 1991). Further verification of the functional disruption ofthe PFI-002 gene may also be made if PFI-002 gene messenger RNA (mRNA)levels and/or PFI-002 polypeptide levels are reduced in cells thatnormally express the PFI-002 gene. Measures of PFI-002 gene mRNA levelsmay be obtained by using reverse transcriptase mediated polymerase chainreaction (RT-PCR), Northern blot analysis, or in situ hybridization. Thequantification of PFI-002 polypeptide levels produced by the cells canbe made, for example, by standard immunoassay methods known in the art.Such immunoassays include but are not limited to, competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme-linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzymatic, or radioisotope labels, for example), Western blots,2-dimensional gel analysis, precipitation reactions, immunofluorescenceassays, protein A assays, and immunoelectrophoresis assays.

Preferred genetically modified animal cells are embryonic stem (ES)cells and ES-like cells. These cells are derived from thepreimplantation embryos and blastocysts of various species, such as mice(Evans et al., Nature 129:154-156, 1981; Martin, Proc. Natl. Acad. Sci.,USA, 78: 7634-7638, 1981), pigs and sheep (Notanianni et al., J. Reprod.Fert. Suppl., 43: 255-260, 1991; Campbell et al., Nature 380:64-68,1996) and primates, including humans (Thomson et al., U.S. Pat.No. 5,843,780, Thomson et al., Science 282: 1145-1147, 1995; and Thomsonet al., Proc. Natl. Acad. Sci. USA 92: 7844-7848, 1995).

These types of cells are pluripotent. That is, under proper conditions,they differentiate into a wide variety of cell types derived from allthree embryonic germ layers: ectoderm, mesoderm and endoderm. Dependingupon the culture conditions, a sample of ES cells can be culturedindefinitely as stem cells, allowed to differentiate into a wide varietyof different cell types within a single sample, or directed todifferentiate into a specific cell type, such as macrophage-like cells,neuronal cells, cardiomyocytes, adipocytes, smooth muscle cells,endothelial cells, skeletal muscle cells, keratinocytes, andhematopoietic cells, such as eosinophils, mast cells, erythroidprogenitor cells, or megakaryocytes. Directed differentiation isaccomplished by including specific growth factors or matrix componentsin the culture conditions, as further described, for example, in Kelleret al., Curr. Opin. Cell Biol. 7: 862-69, 1995, Li et al., Curr. Biol.8: 971, 1998, Klug et al., J. Clin. Invest. 98: 216-24, 1996, Lieschkeet al., Exp. Hematol. 23: 328-34, 1995, Yamane et al., Blood 90:3516-23, 1997, and Hirashima et al., Blood 93: 1253-63, 1999.

The particular embryonic stem cell line that is used for geneticmodification is not critical; exemplary murine ES cell lines includeAB-1 (McMahon and Bradley, Cell 62:1073-85, 1990), E14 (Hooper et al.,Nature 326: 292-95, 1987), D3 (Doetschman et al., J. Embryol. Exp.Morph. 87: 27-45, 1985), CCE (Robertson et al, Nature 323: 445-48,1986), RW4 (Genome Systems, St. Louis, Mo.), and DBA/1 lacJ (Roach etal., Exp. Cell Res. 221: 520-25, 1995).

Production of the Polypeptide

According to the present invention, the production of the polypeptide ofthe present invention can be effected by the culturing of eukaryotic orprokaryotic expression hosts, which have been transformed with one ormore polynucleotides of the present invention, in a conventionalnutrient fermentation medium. The selection of the appropriate mediummay be based on the choice of expression hosts and/or based on theregulatory requirements of the expression construct. Such media arewell-known to those skilled in the art. The medium may, if desired,contain additional components favouring the transformed expression hostsover other potentially contaminating micro-organisms.

Thus, the present invention also provides a method for producing apolypeptide having PFI-002 activity, the method comprising the steps of(a) transforming a host cell with a nucleotide sequence shown in SEQ IDNO: 1 or a derivative, homologue, variant, analogue or fragment thereof;and (b) culturing the transformed host cell under conditions suitablefor the expression of said polypeptide.

The present invention also relates to a method for producing apolypeptide having PFI-002 activity, the method comprising the steps of(a) culturing a host cell that has been transformed with a nucleotidesequence shown in SEQ ID NO: 1 or a derivative, homologue, variant,analogue or fragment thereof under conditions suitable for theexpression of said polypeptide; and (b) recovering said polypeptide fromthe host cell culture.

The present invention also relates to a method for producing apolypeptide having PFI-002 activity, the method comprising the steps of(a) transforming a host cell with a nucleotide sequence shown in SEQ IDNO: 1 or a derivative, homologue, variant, analogue or fragment thereof;(b) culturing the transformed host cell under conditions suitable forthe expression of said polypeptide; and (c) recovering said polypeptidefrom the host cell culture.

Ribozymes

Ribozymes are enzymatic RNA molecules capable of catalysing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridisation of the ribozyme molecule to complementary targetRNA, followed by endonucleolytic cleavage. Within the scope of theinvention are engineered hammerhead motif ribozyme molecules thatspecifically and efficiently catalyse endonucleolytic cleavage ofPFI-002 RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide sequence inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridisation with complementary oligonucleotides usingribonuclease protection assays.

Both antisense RNA and DNA molecules and ribozymes of the invention maybe prepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesisingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro or in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,antisense CDNA constructs that synthesize antisense RNA constitutivelyor inducibly can be introduced into cell lines, cells or tissues.

Detection

The presence of the PFI-002 polynucleotide coding sequence can bedetected by DNA-DNA or DNA-RNA hybridisation or amplification usingprobes, portions or fragments of the sequence presented in SEQ ID NO: 1.Nucleic acid amplification-based assays involve the use ofoligonucleotides or oligomers based on the PFI-002 coding sequence todetect transformants containing PFI-002 DNA or RNA. As used herein“oligonucleotides” or “oligomers” may refer to a nucleic acid sequenceof at least about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20 to25 nucleotides which can be used as a probe or amplimer. Preferably,oligonucleotides are derived from the 3′ region of the nucleotidesequence shown in SEQ ID NO: 1.

A variety of protocols for detecting and measuring the expression ofPFI-002 polypeptide, such as by using either polyclonal or monoclonalantibodies specific for the protein, are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA) and fluorescent activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilising monoclonal antibodies reactive totwo non-interfering epitopes on a PFI-002 polypeptide is preferred, buta competitive binding assay may also be employed. These and other assaysare described, among other places, in Hampton R et al. (1990,Serological Methods, A Laboratory Manual, APS Press, St Paul, Minn.,USA) and Maddox DE et al. (1983, J Exp Med 15 8:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays. Means for producing labelled hybridisation or PCR probes fordetecting PFI-002 polynucleotide sequences include oligolabelling, nicktranslation, end-labelling or PCR amplification using a labellednucleotide. Alternatively, the PFI-002 coding sequence, or any portionof it, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labelled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway, N.J., USA),Promega (Madison, Wis., USA), and US Biochemical Corporation (Cleveland,Ohio, USA) supply commercial kits and protocols for these procedures.Suitable reporter molecules or labels include those radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. No. 3,817,837;U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No.3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149 and U.S.Pat. No. 4,366,241. Also, recombinant immunoglobulins may be produced asshown in U.S. Pat. No. 4,816,567.

Additional methods to quantify the expression of a particular moleculeinclude radiolabelling (Melby P C et al., 1993, J. Immunol Methods Vol159 p235-44) or biotinylating (Duplaa C et al., 1993, Anal Biochem Vol229 p36) nucleotides, co-amplification of a control nucleic acid, andstandard curves onto which the experimental results are interpolated.Quantification of multiple samples may be speeded up by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or calorimetric responsegives rapid quantification.

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression shouldbe confirmed. For example, if the PFI-002 coding sequence is insertedwithin a marker gene sequence, recombinant cells containing PFI-002coding regions can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with a PFI-002coding sequence under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicatesexpression of PFI-002 as well.

Alternatively, host cells which contain the coding sequence for PFI-002and express PFI-002 coding regions may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to,. DNA-DNA or DNA-RNA hybridisation and proteinbioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

Antibodies

The amino acid sequence of the present invention can also be used togenerate antibodies—such as by use of standard techniques—against theamino acid sequence.

Procedures well known in the art may be used for the production ofantibodies to PFI-002 polypeptides. Such antibodies include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, Fabfragments and fragments produced by a Fab expression library.Neutralising antibodies, i.e. those which antagonise biological activityof PFI-002 polypeptides, are especially preferred for diagnostics andtherapeutics.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc. may be immunised by injection with the PFI-002polypeptide or any portion, variant, homologue, fragment, analogue orderivative thereof or oligopeptide which retains immunogenic properties.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminium hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(Bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants which may be employed.

Monoclonal antibodies to the amino acid sequence may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Koehler and Milstein(1975, Nature Vol 256 p495-497), the human B-cell hybridoma technique(Kosbor et al. (1983) Immunol Today Vol 4 p72; Cote et al. (1983)Proceedings of the National Academy of Sciences (USA) Vol 80 p2026-2030)and the EBV-hybridoma technique (Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R Liss Inc, pp. 77-96). In addition,techniques developed for the production of “chimeric antibodies”, thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activitycan be used (Morrison et al. (1984) Proceedings of the National Academyof Sciences (USA) Vol 81 p6851-6855; Neuberger et al. (1984) Nature Vol312 p604-608; Takeda et al. (1985) Nature Vol 314 p452-454).Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,779) can be adapted to producepolypeptide-specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al. (1989, Proceedings of the National Academy of Sciences(USA) Vol 86 p 3833-3837), and Winter G and Milstein C (1991; Nature Vol349 p293-299).

Antibody fragments which contain specific binding sites for PFI-002 mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulphide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W D et al. (1989) Science Vol 256 p1275-1281).

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

PFI-002-specific antibodies are useful for the diagnosis of conditionsand diseases associated with expression of the PFI-002 receptor. Avariety of protocols for competitive binding or immunoradiometric assaysusing either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the formation of complexes between PFI-002 polypeptide and itsspecific antibody (or similar PFI-002-binding molecule) and themeasurement of complex formation. A two-site, monoclonal basedimmunoassay utilising monoclonal antibodies reactive to two,non-interfering epitopes on a specific PFI-002 protein is preferred, buta competitive binding assay may also be employed. These assays aredescribed in Maddox D E et al. (1983, Journal of Experimental MedicineVol 158 p1211).

Anti-PFI-002 antibodies are useful for the diagnosis of disordersinvolving abnormal signal transduction or other disorders or diseasescharacterised by abnormal expression of a PFI-002 receptor. Diagnosticassays for PFI-002 include methods utilising the antibody and a label todetect a PFI-002 polypeptide in human body fluids, cells, tissues orsections or extracts of such tissues. The polypeptides and antibodies ofthe present invention may be used with or without modification.Frequently, the polypeptides and antibodies will be labelled by joiningthem, either covalently or noncovalently, with a reporter molecule. Awide variety of reporter molecules are known to those of skill in theart.

Antibodies may be used in method of detecting polypeptides of theinvention present in biological samples by a method which comprises: (a)providing an antibody of the invention; (b) incubating a biologicalsample with said antibody under conditions which allow for the formationof an antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said antibody is formed.

Antibodies of the invention may be bound to a solid support and/orpackaged into kits in a suitable container along with suitable reagents,controls, instructions and the like.

Assays/Identification Methods

The present invention also relates to an assay method for detecting thepresence of PFI-002 in cells (such as human cells) comprising: (a)performing a reverse transcriptase-polymerase chain reaction (RT-PCR) onRNA (such as total RNA) from such cells using a pair of PCR primers thatare specific for PFI-002, as determined from the DNA sequence shown inSEQ ID NO: 1 or an allelic variation thereof; and (b) assaying theappearance of an appropriately sized PCR fragment—such as by agarose gelelectrophoresis.

There are numerous assays in which the polypeptide of the presentinvention can used to screen for modulators (e.g. antagonists oragonists) of the polypeptide. Examples of such assays include:

Functional Assay—One example of a method for screening receptors toidentify antagonists thereof is to monitor the inhibitory or stimulatoryeffect on cAMP or adenylate cyclase accumulation. Such an assay involvestransfecting a mammalian cell with the receptor of the present inventionfor cell surface expression. The cell is then exposed to putativeantagonists and the amount of cAMP accumulation is measured. If theputative antagonist binds the receptor the levels of receptor-mediatedcAMP or adenylate cyclase activity will either increase or decrease.

Functional Assay using a Fluorometric Imaging Plate Reader (FlipR)—Atechnique used for screening and includes the use of cells that expressthe receptor of the present invention (for example, transfected HEK293cells) in a system that measures intracellular calcium or extracellularpH changes caused by receptor activation. In this technique, cellsexpressing the receptor of the invention may be contacted with compounds(e.g. small molecules, peptides, lipids, nucleotides or glycoproteins)that cause a second messenger response, e.g. signal transduction, changein calcium levels or pH changes. These changes are used to determinewhether the potential compound activates or inhibits the receptor.

Ligand Binding Assay—This type of assay may test binding of a candidatecompound, where adherence to the cells containing the receptor of thepresent invention is detected by means of a label directly or indirectlyassociated with the candidate compound or in an assay involvingcompetition with a labelled competitor. Standard assays for conductingscreens that determine if the compound activates or inhibits thereceptor are well understood by those skilled in the art.

The present invention therefore also relates to a method of identifyingagents (such as compounds, other substances or compositions comprisingthe same) that affect (such as antagonise, agonise or otherwise modify)the activity of PFI-002 and/or the expression thereof, the methodcomprising contacting PFI-002 or the nucleotide sequence coding for thesame with the agent and then measuring the activity of PFI-002 and/orthe expression thereof.

The present invention also relates to a method of identifying agents(such as compounds, other substances or compositions comprising thesame) that selectively affect (such as antagonise, agonise or otherwisemodify) the activity of PFI-002 and/or the expression thereof, themethod comprising contacting PFI-002 or the nucleotide sequence codingfor the same with the agent and then measuring the activity of PFI-002and/or the expression thereof. The present invention also relates to amethod of identifying agents (such as compounds, other substances orcompositions comprising the same) that affect (such as antagonise,agonise or otherwise modify) the activity of PFI-002 and/or theexpression thereof, the method comprising measuring the activity ofPFI-002 and/or the expression thereof in the presence of the agent orafter the addition of the agent in: (a) a cell line into which has beenincorporated recombinant DNA comprising the DNA sequence shown in SEQ IDNO: 1 or an allelic variation thereof, or (b) a cell population or cellline that naturally selectively expresses PFI-002. Preferably, theactivity of PFI-002 is determined by the assay methods described above.

The present invention also relates to a method of identifying agents(such as compounds, other substances or compositions comprising thesame) that selectively affect (such as antagonise, agonise or otherwisemodify) the activity of PFI-002 and/or the expression thereof, themethod comprising measuring the activity of PFI-002 and/or theexpression thereof in the presence of the agent or after the addition ofthe agent in: (a) a cell line into which has been incorporatedrecombinant DNA comprising the DNA sequence shown in SEQ ID NO: 1 or anallelic variation thereof, or (b) a cell population or cell line thatnaturally selectively expresses PFI-002. Preferably, the activity ofPFI-002 is determined by the assay methods described above.

The present invention also relates to a method of screening an agent formodulation (preferably for specific modulation) of PFI-002 (or aderivative, homologue, variant, analogue or fragment thereof) activityor the expression of the nucleotide sequence coding for the same(including a derivative, homologue, variant, analogue or fragmentthereof), the method comprising the steps of: (a) providing a candidateagent; (b) combining PFI-002 (or the derivative, homologue, variant,analogue or fragment thereof) or the nucleotide sequence coding for thesame (or the derivative, homologue, variant, analogue or fragmentthereof) with the candidate agent for a time sufficient to allowmodulation under suitable conditions; and (c) detecting modulation ofthe candidate agent to PFI-002 (or the derivative, homologue, variant,analogue or fragment thereof) or the nucleotide sequence coding for thesame (or the derivative, homologue, variant, analogue or fragmentthereof) in order to ascertain if the candidate agent modulates PFI-002(or the derivative, homologue, variant, analogue or fragment thereofactivity or the expression of the nucleotide sequence coding for thesame (or the derivative, homologue, variant, analogue or fragmentthereof).

The present invention also relates to a method of screening an agent forspecific binding affinity with PFI-002 (or a derivative, homologue,variant, analogue or fragment thereof or the nucleotide sequence codingfor the same (including a derivative, homologue, variant, analogue orfragment thereof), the method comprising the steps of: (a) providing acandidate agent; (b) combining PFI-002 (or the derivative, homologue,variant, analogue or fragment thereof) or the nucleotide sequence codingfor the same (or the derivative, homologue, variant, analogue orfragment thereof) with the candidate agent for a time sufficient toallow binding under suitable conditions; and (c) detecting binding ofthe candidate agent to PFI-002 (or the derivative, homologue, variant,analogue or fragment thereof) or the nucleotide sequence coding for thesame (or the derivative, homologue, variant, analogue or fragmentthereof) in order to ascertain if the candidate agent binds to PFI-002(or the derivative, homologue, variant, analogue or fragment thereof) orthe nucleotide sequence coding for the same (or the derivative,homologue, variant, analogue or fragment thereof).

Thus, in certain embodiments of the present invention, PFI-002 or avariant, homologue, fragment, analogue or derivative thereof and/or acell line that expresses the PFI-002 or variant, homologue, fragment,analogue or derivative thereof may be used to screen for antibodies,peptides, or other agents, such as organic or inorganic molecules, thatact as modulators (e.g. antagonists or agonists) of PFI-002 activity orfor the expression thereof, thereby identifying a therapeutic agentcapable of modulating the receptor. Alternatively, screening of peptidelibraries or organic libraries made by combinatorial chemistry withrecombinantly expressed PFI-002 or a variant, homologue, fragment,analogue or derivative thereof or cell lines expressing PFI-002 or avariant, homologue, fragment, analogue or derivative thereof may beuseful for identification of therapeutic agents that function bymodulating the receptor. Synthetic compounds, natural products, andother sources of potentially biologically active materials can bescreened in a number of ways deemed to be routine to those of skill inthe art. For example, nucleotide sequences encoding the N-terminalregion of PFI-002 may be expressed in a cell line, which can be used forscreening of allosteric modulators, either agonists or antagonists, ofPFI-002 activity.

A PFI-002 polypeptide, its immunogenic fragments or oligopeptidesthereof can be used for screening therapeutic compounds in any of avariety of drug screening techniques. The polypeptide employed in such atest may be free in solution, affixed to a solid support, borne on acell surface, or located intracellularly. The formation of bindingcomplexes between a PFI-002 polypeptide and the agent being tested maybe measured.

Accordingly, the present invention relates to a method for screening oneor a plurality of compounds for modulation (preferably specificmodulation, such as specific binding affinity) of PFI-002 or theexpression thereof, or a portion thereof or variant, homologue,fragment, analogue or derivative thereof, comprising providing one or aplurality of compounds; combining a PFI-002 or a nucleotide sequencecoding for the same or a portion thereof or variant, homologue,fragment, analogue or derivative thereof with the or each of a pluralityof compounds for a time sufficient to allow modulation under suitableconditions; and detecting binding of a PFI-002, or portion thereof orvariant, homologue, fragment, analogue or derivative thereof, to each ofthe plurality of compounds, thereby identifying the compound orcompounds which modulate a PFI-002 or a nucleotide sequence coding forthe same. In such an assay, the plurality of compounds may be producedby combinatorial chemistry techniques known to those of skill in theart.

Another technique for drug screening provides for high throughputscreening (HTS) of compounds having suitable binding affinity to thePFI-002 polypeptides and is based upon the method described in detail inGeysen, WO 84/03564, published on Sep. 13, 1984. In summary, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with PFI-002 fragments and washed. A boundPFI-002 is then detected—such as by appropriately adapting methods wellknown in the art. A purified PFI-002 can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralising antibodies can be used to capture thepeptide and immobilise it on a solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralising antibodies capable of binding a PFI-002polypeptide specifically compete with a test compound for binding aPFI-002. In this manner, the antibodies can be used to detect thepresence of any peptide which shares one or more antigenic determinantswith a PFI-002.

The assay method of the present invention may be a high throughputscreen (HTS). In this regard, the teachings of WO 84/03564 may beadapted for the PFI-002 of the present invention.

The teachings of U.S. Pat. No. 5,738,985 may also be adapted for theassay method of the present invention.

Agents

The present invention also provides one or more agents identified by theassays methods and identification methods of the present invention.

The agent of the present invention can be, for example, an organiccompound or an inorganic compound. The agent can be, for example, anucleotide sequence that is antisense to all or part of the sequenceshown in SEQ ID NO: 1.

The invention further provides an agent of the present invention (oreven a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable solvate thereof) or a pharmaceutical composition containingany of the foregoing, for use as a medicament.

The present invention also relates to the use of an agent to affectPFI-002 activity (such as to antagonise, modulate or agonise its GPCRactivity).

Diagnostics

The present invention also provides a diagnostic composition for thedetection of PFI-002 polynucleotide sequences. The diagnosticcomposition may comprise the sequence shown in SEQ ID NO: 1 or avariant, homologue, fragment, analogue or derivative thereof, or asequence capable of hybridising to all or part of the nucleotidesequence shown in SEQ ID NO: 1 or an allelic variation thereof.

In order to provide a basis for the diagnosis of disease, normal orstandard values from a PFI-002 polypeptide expression should beestablished. This is accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, withantibody to a PFI-002 polypeptide under conditions suitable for complexformation which are well known in the art. The amount of standardcomplex formation may be quantified by comparing it to a dilution seriesof positive controls where a known amount of antibody is combined withknown concentrations of a purified PFI-002 polypeptide. Then, standardvalues obtained from normal samples may be compared with values obtainedfrom samples from subjects potentially affected by a disorder or diseaserelated to a PFI-002 polypeptide expression. Deviation between standardand subject values establishes the presence of the disease state.

A PFI-002 polynucleotide, or any part thereof, may provide the basis fora diagnostic and/or a therapeutic compound. For diagnostic purposes,PFI-002 polynucleotide sequences may be used to detect and quantify geneexpression in conditions, disorders or diseases in which PFI-002activity may be implicated.

PFI-002-encoding polynucleotide sequence may be used for the diagnosisof diseases resulting from expression of PFI-002. For example,polynucleotide sequences encoding PFI-002 may be used in hybridisationor PCR assays of tissues from biopsies or autopsies or biologicalfluids, such as serum, synovial fluid or tumour biopsy, to detectabnormalities in PFI-002 expression. The form of such qualitative orquantitative methods may include Southern or northern analysis, dot blotor other membrane-based technologies; PCR technologies; dip stick, pinor chip technologies; and ELISA or other multiple sample formattechnologies. All of these techniques are well known in the art and are,in fact, the basis of many commercially available diagnostic kits.

Such assays may be tailored to evaluate the efficacy of a particulartherapeutic treatment regime and may be used in animal studies, inclinical trials, or in monitoring the treatment of an individualpatient. In order to provide a basis for the diagnosis of disease, anormal or standard profile for PFI-002 expression should be established.This is accomplished by combining body fluids or cell extracts takenfrom normal subjects, either animal or human, with PFI-002 or a portionthereof, under conditions suitable for hybridisation or amplification.Standard hybridisation may be quantified by comparing the valuesobtained for normal subjects with a dilution series of positive controlsrun in the same experiment where a known amount of purified PFI-002 isused. Standard values obtained from normal samples may be compared withvalues obtained from samples from subjects potentially affected by adisorder or disease related to expression of the PFI-002 codingsequence. Deviation between standard and subject values establishes thepresence of the disease state. If disease is established, an existingtherapeutic agent is administered, and treatment profile or values maybe generated. Finally, the assay may be repeated on a regular basis toevaluate whether the values progress toward or return to the normal orstandard pattern. Successive treatment profiles may be used to show theefficacy of treatment over a period of several days or several months.

Thus, the present invention relates to the use of a PFI-002 polypeptide,or variant, homologue, fragment, analogue or derivative thereof, toproduce anti-PFI-002 antibodies which can, for example, be useddiagnostically to detect and quantify PFI-002 levels in disease states.

The present invention further relates to diagnostic assays and kits forthe detection of PFI-002 in cells and tissues comprising a purifiedPFI-002 which may be used as a positive control, and anti-PFI-002antibodies. Such antibodies may be used in solution-based,membrane-based, or tissue-based technologies to detect any disease stateor condition related to the expression of PFI-002 protein or expressionof deletions or a variant, homologue, fragment, analogue or derivativethereof.

Probes

Another aspect of the subject invention is the provision of nucleic acidhybridisation or PCR probes which are capable of detectingpolynucleotide sequences, including genomic sequences, encoding PFI-002coding region or closely related molecules, such as alleles. Thespecificity of the probe, i.e. whether it is derived from a highlyconserved, conserved or non-conserved region or domain, and thestringency of the hybridisation or amplification (high, intermediate orlow) will determine whether the probe identifies only naturallyoccurring PFI-002 coding sequence, or related sequences. Probes for thedetection of related nucleic acid sequences are selected from conservedor highly conserved nucleotide regions of PFI-002 polynucleotides, suchas the 3′ region, and such probes may be used in a pool of degenerateprobes. For the detection of identical nucleic acid sequences, or wheremaximum specificity is desired, nucleic acid probes are selected fromthe non-conserved nucleotide regions or unique regions of PFI-002polynucleotides. As used herein, the term “non-conserved nucleotideregion” refers to a nucleotide region that is unique to the PFI-002coding sequence disclosed herein and does not occur in relatedsequences.

PCR, as described in U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,800,195and U.S. Pat. No. 4,965,188 provides additional uses foroligonucleotides based upon the PFI-002 sequence. Such oligomers aregenerally chemically synthesized, but they may be generatedenzymatically or produced from a recombinant source. Oligomers generallycomprise two nucleotide sequences, one with sense orientation (5′→3′)and one with antisense (3′←5′) employed under optimised conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantification of closely related DNA or RNA sequences.

The nucleic acid sequence for PFI-002 can also be used to generatehybridisation probes as previously described, for mapping the endogenousgenomic sequence. The sequence may be mapped to a particular chromosomeor to a specific region of the chromosome using well known techniques.These include in situ hybridisation to chromosomal spreads (Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York City, USA), flow-sorted chromosomal preparations, or artificialchromosome constructions such as yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial PI constructions orsingle chromosome cDNA libraries.

In situ hybridisation of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers are invaluable in extending genetic maps. Examples of geneticmaps can be found in Science (1995; 270:410f and 1994; 265:1981f). Oftenthe placement of a gene on the chromosome of another mammalian speciesmay reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once a disease orsyndrome, such as ataxia telangiectasia (AT), has been crudely localisedby genetic linkage to a particular genomic region, for example, AT to11q22-23 (Gatti et al (1988) Nature 336:577-580), any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation. The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc. between normal, carrier or affectedindividuals.

Pharmaceuticals

The present invention also provides a pharmaceutical composition fortreating an individual in need of the same due to PFI-002 activity, thecomposition comprising a therapeutically effective amount of an agentthat modulates (such as antagonises or agonises) said activity and apharmaceutically acceptable carrier, diluent, excipient or adjuvant.

Thus, the present invention also covers pharmaceutical compositionscomprising the agents of the present invention (an agent capable ofmodulating the expression pattern of the nucleotide sequence of thepresent invention or the activity of the expression product thereofand/or an agent identified by an assay according to the presentinvention). In this regard, and in particular for human therapy, eventhough the agents of the present invention can be administered alone,they will generally be administered in admixture with a pharmaceuticalcarrier, adjuvant, excipient or diluent selected with regard to theintended route of administration and standard pharmaceutical practice.

By way of example, in the pharmaceutical compositions of the presentinvention, the agents of the present invention may be admixed with anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),or solubilising agent(s).

In general, a therapeutically effective daily oral or intravenous doseof the agents of the present invention is likely to range from 0.01 to50 mg/kg body weight of the subject to be treated, preferably 0.1 to 20mg/kg. The agents of the present invention may also be administered byintravenous infusion, at a dose which is likely to range from 0.001-10mg/kg/hr.

Thus, the present invention also provides a method of treating anindividual in need of the same due to PFI-002 activity comprisingadministering to said individual an effective amount of thepharmaceutical composition of the present invention.

Typically, the physician will determine the actual dosage which will bemost suitable for an individual patient and it will vary with the age,weight, sex and response of the particular patient. The above dosagesare exemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intracavernosally, intravenously,intramuscularly or subcutaneously. For parenteral administration, thecompositions may be best used in the form of a sterile aqueous solutionwhich may contain other substances, for example enough salts ormonosaccharides to make the solution isotonic with blood. For buccal orsublingual administration the compositions may be administered in theform of tablets or lozenges which can be formulated in a conventionalmanner.

For oral, parenteral, buccal and sublingual administration to subjects(such as patients), the daily dosage level of the agents of the presentinvention may typically be from 10 to 500 mg (in single or divideddoses). Thus, and by way of example, tablets or capsules may containfrom 5 to 100 mg of active agent for administration singly, or two ormore at a time, as appropriate. It is also possible to administer theagents of the present invention in sustained release formulations.

In some applications, generally in humans, oral administration of theagents of the present invention is the preferred route, being the mostconvenient and can in some cases avoid disadvantages associated withother routes of administration—such as those associated withintracavernosal (i.c.) administration. In circumstances where therecipient suffers from a swallowing disorder or from impairment of drugabsorption after oral administration, the drug may be administeredparenterally, sublingually or buccally.

For veterinary use, the agent of the present invention is typicallyadministered as a suitably acceptable formulation in accordance withnormal veterinary practice and the veterinary surgeon will determine thedosing regimen and route of administration which will be mostappropriate for a particular animal. However, as with human treatments,it may be possible to administer the agent alone for veterinarytreatments.

Typically, the pharmaceutical compositions—which may be for human oranimal usage—will comprise any one or more of a pharmaceuticallyacceptable diluent, carrier, excipient or adjuvant. The choice ofpharmaceutical carrier, excipient, adjuvant or diluent can be selectedwith regard to the intended route of administration and standardpharmaceutical practice. As indicated above, the pharmaceuticalcompositions may comprise as—or in addition to—the carrier, excipient,adjuvant or diluent any suitable binder(s), lubricant(s), suspendingagent(s), coating agent(s) or solubilising agent(s).

In some embodiments of the present invention, the pharmaceuticalcompositions will comprise one or more of: an agent that has beenscreened by an assay of the present invention; an agent that is capableof interacting with SEQ ID NO: 1 or SEQ ID NO: 2 including derivatives,fragments, homologues, analogues or variants thereof or sequencescapable of hybridising to the nucleotide sequence shown in SEQ ID NO: 1.

Included in the scope of the invention are oligonucleotide sequences,antisense RNA and DNA molecules and ribozymes, which function todestabilise PFI-002 mRNA or inhibit translation of PFI-002.

A PFI-002 antisense molecule may provide the basis for treatment ofvarious abnormal conditions related to, for example, increased PFI-002activity.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of recombinant PFI-002 sense or antisense molecules to thetargeted cell population. Methods which are well known to those skilledin the art can be used to construct recombinant vectors containingPFI-002. Alternatively, recombinant PFI-002 can be delivered to targetcells in liposomes.

The full length cDNA sequence and/or its regulatory elements enableresearchers to use PFI-002 as a tool in sense (Youssoufian H and H FLodish (1993) Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991)Annu Rev Biochem 60:631-652) investigations of gene function.Oligonucleotides, designed from the cDNA or control sequences obtainedfrom the genomic DNA can be used in vitro or in vivo to inhibitexpression. Such technology is now well known in the art, and sense orantisense oligonucleotides or larger fragments can be designed fromvarious locations along the coding or control regions. Appropriateoligonucleotides, which can be 20 nucleotides in length, may be used toisolate PFI-002 sequences or closely related molecules from humanlibraries.

Additionally, PFI-002 expression can be modulated by transfecting a cellor tissue with expression vectors which express high levels of a PFI-002fragment in conditions where it would be preferable to block PFI-002activity. Such constructs can flood cells with untranslatable sense orantisense sequences. Even in the absence of integration into the DNA,such vectors may continue to transcribe RNA molecules until all copiesof the vector are disabled by endogenous nucleases. Such transientexpression may last for a month or more with a non-replicating vectorand even longer if appropriate replication elements are part of thevector system.

Modifications of gene expression can be obtained by designing antisensesequences to the control regions of the PFI-002 gene, such as thepromoters, enhancers, and introns.

Oligonucleotides derived from the transcription initiation site, e.g.between −10 and +10 regions of the leader sequence, are preferred.Antisense RNA and DNA molecules may also be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes. Similarly, inhibition can be achieved using Hogeboombase-pairing methodology, also known as “triple helix” base-pairing.Triple helix pairing compromises the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules.

Thus the invention provides a pharmaceutical composition comprising anagent of the present invention (or even a pharmaceutically acceptablesalt thereof, or a pharmaceutically acceptable solvate thereof) togetherwith a pharmaceutically acceptable diluent, adjuvant, excipient orcarrier.

The pharmaceutical composition could be for veterinary (i.e. animal)usage or for human usage.

Thus, the present invention therefore also relates to pharmaceuticalcompositions comprising effective amounts of modulators (e.g.antagonists or agonists) of PFI-002 protein (including antisense nucleicacid sequences) in admixture with a pharmaceutically acceptable diluent,carrier, excipient or adjuvant (including combinations thereof).

The present invention relates to pharmaceutical compositions which maycomprise all or portions of PFI-002 polynucleotide sequences, PFI-002antisense molecules, PFI-002 polypeptides, protein, peptide or organicmodulators of PFI-002 bioactivity, such as antagonists (includingantibodies) or agonists, alone or in combination with at least one otheragent, such as stabilising compound, and may be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water.

General Methodology References

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., ShortProtocols in Molecular Biology (1999) 4^(th) Ed, John Wiley & Sons, Inc.PCR is described in U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,800,195 andU.S. Pat. No. 4,965,188.

Deposits

The following sample was deposited in accordance with the BudapestTreaty at the recognised depositary The National Collections ofIndustrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive,Aberdeen, Scotland, AB2 1RY, United Kingdom on 23 Aug. 2000:

NCIMB number NCIMB 41066 is Escherichia coli Pfi-002.

The depositor was Pfizer Central Research, Pfizer Limited, RamsgateRoad, Sandwich, Kent, CT13 9N.J., United Kingdom.

One skilled in the art could readily grow the above-mentioned E. coliclone (NCIMB 41066) in Luria Broth containing ampicillin and isolate theplasmid DNA of the clone using the alkali lysis method as described inSambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, N.Y., USA. The di-deoxytermination method as described by Sanger et al. (Proceedings of theNational Academy of Sciences (USA) (December 1977), 74(12):5463-5467)and modified by Applied Biosystems (see Applied Biosystemsmanufacturer's literature) for fluorescent detection could then be usedto sequence the DNA and identify PFI-002.

The present invention also encompasses sequences derivable and/orexpressable from that deposit and embodiments comprising the same. Thepresent invention also encompasses partial sequences derivable and/orexpressable from that deposit and embodiments comprising the same,wherein those partial sequences code for active polypeptides. Thepresent invention also encompasses proteins comprising sequencesderivable and/or expressable from that deposit and embodimentscomprising the same. The present invention also encompasses proteinscomprising partial sequences derivable and/or expressable from thatdeposit and embodiments comprising the same, wherein those partialsequences code for active polypeptides.

INTRODUCTION TO THE EXAMPLES SECTION, THE FIGURES AND THE SEQUENCELISTING

The polynucleotide which encodes the GPCR of the present invention wascloned and the DNA and amino acid sequences analysed using variousbioinformatic tools. The GPCR encoded by the sequences described hereinhas been termed PFI-002. The present invention will now be described, byway of example only, with reference to the accompanying Figures andSequence Listing Examples

Identification of PFI-002

PFI-002 was identified in unannotated genomic sequence from chromosome 5(GenBank contig AC008571) which was released by the Genome SequencingCenters by searching the sequences with known members of the G-proteincoupled receptor (GPCR) family using the BLAST algorithm.

PFI-002 sequence was found amongst the following public and Incyte (USA)expressed sequence tags (ESTs): Public ESTs g1186640 Nervous Systemyy59b04 multiple sclerosis g766966 Nervous System yf62a02 brain, infantg876179 Nervous System ym13d04 brain, infant Incyte ESTs 621183H1Endocrine System 621183 paraganglion tumor

This suggests that the PFI-002 gene is expressed in the nervous systemand possibly tumour cells.

Bioinformatic Studies

In order to confirm that PFI-002 was a member of the GPCR family, anumber of bioinformatics approaches were performed.

(a) BLAST Search Against Swissprot

PFI-002 was searched against Swissprot using the BLAST algorithm (BasicLocal Alignment Search Tool (Altshul S F (1993) J.Mol. Evol. 36:290-300;Altshul, S F et al (1990) J. Mol. Biol. 215:403-410)) to identify theclosest protein match. In this case the top hit was to:

-   -   SW|P20789INTR1_RAT NEUROTENSIN RECEPTOR TYPE 1 (NT-R-1) (HIGH-A        . . . )

These results indicate that PFI-002 is a member of the GPCR family.

(b) ClustalW Alignment of PFI-002 with SW|P20789INTR1_RAT NEUROTENSINRECEPTOR TYPE 1 (NT-R-1)

These results are shown in FIG. 3.

(c) BLAST Search Against a Non-redundant Human GPCR Database

PFI-002 was searched against a non-redundant human GPCR databasecomprising mainly sequences from Genbank and Geneseq Patents databasesin order to identify the class of agonist for this receptor. The top tenhits are shown below: AF044601 NMUR1 : e-value = 5e−73, % Identity = 56%AF034632 MOTILIN : e-value = 1e−31, % Identity = 37% P30989 NTR1 :e-value = 2e−30, % Identity = 35% U60179 GHSR : e-value = 3e−25, %Identity = 32% Y10148 NTR2 : e-value = 4e−24, % Identity = 31% P16473TRFR : e-value = 4e−23, % Identity = 33% P30874 SSR2 : e-value = 4e−22,% Identity = 31% P35372 OPRM : e-value = 7e−22, % Identity = 31% P30556AT1B : e-value = 6e−21, % Identity = 31% L08893 BRS3 : e-value = 8e−21,% Identity = 26%.(e value = statistical likelihood of the hit occurring by chance)

These results demonstrate that PFI-002 is most closely similar toneuromedin U receptors and they suggest that PFI-002 encodes a novelGPCR whose ligand is likely to be neuromedin U.

(d) ClustalW Alignment of PFI-002 with GB|AF044601|NMUR1_HumanNEUROMEDIN RECEPTOR TYPE 1 (NMU-R-1)

These results are shown in FIG. 4.

Isolation of PFI-002

Full-length coding sequence of PFI-002 was cloned from colon poly A⁺RNA, using the thermoscript reverse transcriptase-polymerase chainreaction (RT-PCR) system (Life Technologies, Inc.) according to themanufacturer's instructions.

Reverse transcription: The 3′-5′ gene specific primer used in the RTreaction was: GSP1: 5′- GCTCTGAAAGAATTCAGGTTTTG-3′ (SEQ ID NO: 4)

RT reaction: 500 ng human colon poly A⁺ RNA, 1 μl GSP1 (10 μM stock),made up to 10 μl with H₂O. Proceeded as detailed in the manufacturer'sinstructions.

PCR: 2 μl of the RT reaction was used per PCR reaction following themanufacturer's protocol. 5′-3′ primer: GSP25′-ACCATGGCAGGGATGGAAAAACTT-3′ (SEQ ID NO: 3) 3′-5′ primer: GSP15′-GCTCTGAAAGAATTCAGGTTTTG-3′ (SEQ ID NO: 4)

PCR conditions: 94° C.—2 minutes, then 30 cycles of 94° C.—1 minute, 58°C.—1 minute, 68° C.—2 minutes. Final cycle was 68° C.—14 minutes.

The PFI-002 PCR product was gel extracted using the QIAgen gelextraction kit, according to the manufacturer's instructions. Theresultant product was TA cloned (Invitrogen TA cloning methodology) intothe vector pcDNA3.1N5-His-TOPO (Invitrogen), according to themanufacturer's instructions.

Tissue Distribution Forward Primer A1: 5′-TCCAGAAACACCTGAACAGC-3′ (SEQID NO: 5) Reverse Primer A2: 5′-GAGGTAGAATAGGAAGGAGG-3′ (SEQ ID NO: 6)

Expected product size=615 bp

PCR carried out on 250 ng of human tissue cDNA over 40 cycles.

PCR reaction set up as follows: dNTPs (10 mM)—1 μl, Primer A1 (10 μM)—1μl, Primer A2 (10 μM)—1 μl, 5× reaction buffer—10 μl, Elongase (LifeTechnologies, Inc.)—1 μl, cDNA—250 μg, made up to 50 μl with H₂0.

Results Tissue PFI-002 Tissue PFI-002 Fetal Brain − Fetal heart −Cerebellum +++ Liver − Dorsal Root − Lung ++ Ganglia Bladder − Hela −Heart − Stomach +++ Kidney ++ Small intestine − Fetal kidney +++ Colon +Testis +++ Spleen −Key to Table:−: not detected,+: low expression,+++: high expressionFunctional StudiesTransient Expression in Native HEK 293 Cells

Transfection: 7.5 μg of PFI-002 DNA was transiently transfected intonative human embryonic kidney 293 (HEK 293) cells (75 cm² flask, 80%confluent) using the Lipofectamine Plus protocol (Life Technologies,Inc.) and following the manufacturer's instructions. 24 hrs aftertransfection, the cells were detached with trypsin and seeded into blackwalled 96 well plates at a density of 5×10⁴ cells/well. The next day thecells were loaded with Fluo-3 in the presence of 3 mM probenicid(Molecular Devices; inhibits activity of the anion transport protein,thus improving dye loading) for 1.5 hours (loading dye/96 well plate: 50μg Fluo-3 in 20 μl DMSO+20 , 20% pluronic acid in DMSO+11 ml growthmedia +3 mM probenicid—added 100 μl loading dye/well). After washing(wash buffer: PBS/3 mM probenicid/pH 7.4), the cells were exposed to 60peptides (final concentration 5 μM each) arrayed in a 96 well plate. Inaddition, neuromedins B, C, N, U8 and U25 (final concentration 10 μMeach) were screened in triplicate. Fluorescence was measured by aFluorometric Imaging Plate Reader (FLIPR, Molecular Devices) accordingto the manufacturer's protocol.

Screened Peptides:

Location on 96 Well Plate Peptide (see FIG. 2)

-   -   A1 Arg-Gly-Asp-Ser (RGDS)    -   A2 Adrenocorticotrophic hormone (1-39), human (ACTH)    -   A3 Pituitary adenylate cyclase activating peptide (1-27)    -   A4 Neuropeptide Y, human, rat (NPY)    -   A5 Guanylin (rat)    -   A6 alpha-Calcitonin gene-related peptide, human (CGRP)    -   A7 beta-Melanocyte stimulating hormone, human (beta-MSH)    -   A8 Melanin-concentrating cormone, human, rat, mouse (MCH)    -   A9 Somatostatin-14    -   A10 Inhibin beta-subunit fragment (67-94), human    -   A11 Thyrotropin-releasing hormone (TRH)    -   A12 Angiotensin II, human    -   B1 [Arg8]Vasopressin    -   B2 [Arg8]Vasotocin (AVT)    -   B3 Substance P    -   B4 Bradykinin    -   B5 Neurokinin B    -   B6 Cholecystokinin sulfated (CCK-8 sulfated)    -   B7 Bombesin    -   B8 Calcitonin, human    -   B9 Oxytocin    -   B10 Dynorphin A    -   B11 beta-Endorphin, human    -   B12 Leu-enkephalin    -   C1 Met-enkephalin    -   C2 Brain natriuretic peptide-3.2, porcine    -   C3 N-Formyl-Met-Leu-Phe    -   C4 Nociceptin (Orphanin FQ)    -   C5 Urodilatin    -   C6 Gastrin I, human    -   C7 Neuromedin B    -   C8 Urocortin, rat    -   C9 Leptin fragment (22-56), human    -   C10 Neurokinin A    -   C11 Vasoactive intestinal peptide, human, bovine, porcine, rat        (VIP)    -   C12 Secretin, human    -   D1 Brain injury-derived neurotrophic peptide (BINP)    -   D2 Endomorphin-1    -   D3 Endomorphin-2    -   D4 Luteinizing hormone releasing hormone (LHRH)    -   D5 Neuromedin N, porcine    -   D6 Nocistatin, bovine    -   D7 Motilin, porcine    -   D8 gamma-Melanocyte stimulating hormone (gamma-MSH)    -   D9 Neurotensin    -   D10 Parathyroid hormone (1-34), human    -   D11 Sauvagine, frog    -   D12 Valosin, porcine    -   E1 Neuropeptide FF (F-8-F-NH2)    -   E2 Peptide histidine methionine-27, human (PHM-27)    -   E3 Enterostatin, human    -   E4 Gastrin-releasing peptide, human    -   E5 beta-Amyloid peptide (1-28)    -   E6 CRF (human, rat)    -   E7 Galanin, human    -   E8 Kinetensin, human, rat    -   E9 Antiflammin-1    -   E10 Antiflammin-2    -   E11 Mastoparan, wasp    -   E12 Peptide YY, human    -   F1 Neuromedin B    -   F2 Neuromedin B    -   F3 Neuromedin B    -   F4 Neuromedin C    -   F5 Neuromedin C    -   F6 Neuromedin C    -   F7 Neuromedin N    -   F8 Neuromedin N    -   F9 Neuromedin N    -   F10 Neuromedin U8    -   F11 Neuromedin U8    -   F12 Neuromedin U8    -   G1 Neuromedin U25    -   G2 Neuromedin U25    -   G3 Neuromedin U25

The results of the screen can be seen in FIG. 2, which indicates thespecific functional response of PFI-002 receptor to neuromedin U. Allother responses seen in FIG. 2 are the result of endogenously expressedreceptors which are native to the parental cell line HEK 293.

It will be appreciated that the foregoing is provided by way of exampleonly and modification of detail may be made without departing from thescope of the invention.

1-12. (Cancelled).
 13. A polypeptide comprising: (a) an amino acidsequence encoded by a polynucleotide sequence of SEQ ID NO: 1; (b) theamino acid sequence of SEQ ID NO: 2; (c) an amino acid sequence encodedby the cDNA contained in the clone deposited as National Collections ofIndustrial and Marine Bacteria Limited (NCIMB) 41066; or (d) an aminoacid sequence encoded by a polynucleotide that has at least 95% identityto the nucleotide sequence of SEQ ID NO: 1 and encodes a neuromedin Ureceptor that mediates, in HEK-293 cells transfected with and expressingsaid polynucleotide, a specific neuromedin U-induced increase inintracellular calcium following contact with neuromedin U.
 14. Anantibody against the polypeptide of claim
 13. 15. (Cancelled).
 16. Apharmaceutical composition comprising the antibody of claim 14 and oneor more pharmaceutically acceptable carriers, diluents, adjuvants orexcipients. 17-22. (Cancelled).